Preparing Better Samples for Cryo-Electron Microscopy: Biochemical Challenges Do Not End with Isolation and Purification.

The preparation of extremely thin samples, which are required for high-resolution electron microscopy, poses extreme risk of damaging biological macromolecules due to interactions with the air-water interface. Although the rapid increase in the number of published structures initially gave little indication that this was a problem, the search for methods that substantially mitigate this hazard is now intensifying. The two main approaches under investigation are (a) immobilizing particles onto structure-friendly support films and (b) reducing the length of time during which such interactions may occur. While there is little possibility of outrunning diffusion to the interface, intentional passivation of the interface may slow the process of adsorption and denaturation. In addition, growing attention is being given to gaining more effective control of the thickness of the sample prior to vitrification.

A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation.

Many organisms evolved strategies to survive desiccation. Plant seeds protect dehydrated embryos from various stressors and can lay dormant for millennia. Hydration is the key trigger to initiate germination, but the mechanism by which seeds sense water remains unresolved. We identified an uncharacterized Arabidopsis thaliana prion-like protein we named FLOE1, which phase separates upon hydration and allows the embryo to sense water stress. We demonstrate that biophysical states of FLOE1 condensates modulate its biological function in vivo in suppressing seed germination under unfavorable environments. We find intragenic, intraspecific, and interspecific natural variation in FLOE1 expression and phase separation and show that intragenic variation is associated with adaptive germination strategies in natural populations. This combination of molecular, organismal, and ecological studies uncovers FLOE1 as a tunable environmental sensor with direct implications for the design of drought-resistant crops, in the face of climate change.

African lungfish genome sheds light on the vertebrate water-to-land transition.

Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.

Current Understanding of the Mechanism of Water Oxidation in Photosystem II and Its Relation to XFEL Data.

The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.

Developmental Responses to Water and Salinity in Root Systems.

Roots provide the primary mechanism that plants use to absorb water and nutrients from their environment. These functions are dependent on developmental mechanisms that direct root growth and branching into regions of soil where these resources are relatively abundant. Water is the most limiting factor for plant growth, and its availability is determined by the weather, soil structure, and salinity. In this review, we define the developmental pathways that regulate the direction of growth and branching pattern of the root system, which together determine the expanse of soil from which a plant can access water. The ability of plants to regulate development in response to the spatial distribution of water is a focus of many recent studies and provides a model for understanding how biological systems utilize positional cues to affect signaling and morphogenesis. A better understanding of these processes will inform approaches to improve crop water use efficiency to more sustainably feed a growing population.

A Potassium-Dependent Oxygen Sensing Pathway Regulates Plant Root Hydraulics.

Aerobic organisms survive low oxygen (O2) through activation of diverse molecular, metabolic, and physiological responses. In most plants, root water permeability (in other words, hydraulic conductivity, Lpr) is downregulated under O2 deficiency. Here, we used a quantitative genetics approach in Arabidopsis to clone Hydraulic Conductivity of Root 1 (HCR1), a Raf-like MAPKKK that negatively controls Lpr. HCR1 accumulates and is functional under combined O2 limitation and potassium (K(+)) sufficiency. HCR1 regulates Lpr and hypoxia responsive genes, through the control of RAP2.12, a key transcriptional regulator of the core anaerobic response. A substantial variation of HCR1 in regulating Lpr is observed at the Arabidopsis species level. Thus, by combinatorially integrating two soil signals, K(+) and O2 availability, HCR1 modulates the resilience of plants to multiple flooding scenarios.

The CTPase activity of ParB determines the size and dynamics of prokaryotic DNA partition complexes.

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.

Understanding Fungi in Glacial and Hypersaline Environments.

Hypersaline waters and glacial ice are inhospitable environments that have low water activity and high concentrations of osmolytes. They are inhabited by diverse microbial communities, of which extremotolerant and extremophilic fungi are essential components. Some fungi are specialized in only one of these two environments and can thrive in conditions that are lethal to most other life-forms. Others are generalists, highly adaptable species that occur in both environments and tolerate a wide range of extremes. Both groups efficiently balance cellular osmotic pressure and ion concentration, stabilize cell membranes, remodel cell walls, and neutralize intracellular oxidative stress. Some species use unusual reproductive strategies. Further investigation of these adaptations with new methods and carefully designed experiments under ecologically relevant conditions will help predict the role of fungi in hypersaline and glacial environments affected by climate change, decipher their stress resistance mechanisms and exploit their biotechnological potential.

Renal Autocrine and Paracrine Signaling: A Story of Self-protection.

Autocrine and paracrine signaling in the kidney adds an extra level of diversity and complexity to renal physiology. The extensive scientific production on the topic precludes easy understanding of the fundamental purpose of the vast number of molecules and systems that influence the renal function. This systematic review provides the broader pen strokes for a collected image of renal paracrine signaling. First, we recapitulate the essence of each paracrine system one by one. Thereafter the single components are merged into an overarching physiological concept. The presented survey shows that despite the diversity in the web of paracrine factors, the collected effect on renal function may not be complicated after all. In essence, paracrine activation provides an intelligent system that perceives minor perturbations and reacts with a coordinated and integrated tissue response that relieves the work load from the renal epithelia and favors diuresis and natriuresis. We suggest that the overall function of paracrine signaling is reno-protection and argue that renal paracrine signaling and self-regulation are two sides of the same coin. Thus local paracrine signaling is an intrinsic function of the kidney, and the overall renal effect of changes in blood pressure, volume load, and systemic hormones will always be tinted by its paracrine status.

Ancestral desiccation tolerance tools repurposed throughout plant evolution.

The ability to tolerate and recover from desiccation is an adaptation that permitted primitive plants to colonize land, and it persists in select species today. Zhang et al. dissected desiccation tolerance in moss species, and traced a key regulator through evolution to identify a conserved mechanism of water sensing in angiosperms.

Multidomain Convergence of Argonaute during RISC Assembly Correlates with the Formation of Internal Water Clusters.

Despite the relevance of Argonaute proteins in RNA silencing, little is known about the structural steps of small RNA loading to form RNA-induced silencing complexes (RISCs). We report the 1.9 Å crystal structure of human Argonaute4 with guide RNA. Comparison with the previously determined apo structure of Neurospora crassa QDE2 revealed that the PIWI domain has two subdomains. Binding of guide RNA fastens the subdomains, thereby rearranging the active-site residues and increasing the affinity for TNRC6 proteins. We also identified two water pockets beneath the nucleic acid-binding channel that appeared to stabilize the mature RISC. Indeed, mutating the water-pocket residues of Argonaute2 and Argonaute4 compromised RISC assembly. Simulations predict that internal water molecules are exchangeable with the bulk solvent but always occupy specific positions at the domain interfaces. These results suggest that after guide RNA-driven conformational changes, water-mediated hydrogen-bonding networks tie together the converged domains to complete the functional RISC structure.

Understanding the Asian water tower requires a redesigned precipitation observation strategy.

The Asian water tower (AWT) serves as the source of 10 major Asian river systems and supports the lives of ~2 billion people. Obtaining reliable precipitation data over the AWT is a prerequisite for understanding the water cycle within this pivotal region. Here, we quantitatively reveal that the "observed" precipitation over the AWT is considerably underestimated in view of observational evidence from three water cycle components, namely, evapotranspiration, runoff, and accumulated snow. We found that three paradoxes appear if the so-called observed precipitation is corrected, namely, actual evapotranspiration exceeding precipitation, unrealistically high runoff coefficients, and accumulated snow water equivalent exceeding contemporaneous precipitation. We then explain the cause of precipitation underestimation from instrumental error caused by wind-induced gauge undercatch and the representativeness error caused by sparse-uneven gauge density and the complexity of local surface conditions. These findings require us to rethink previous results concerning the water cycle, prompting the study to discuss potential solutions.

Continuous ammonia synthesis from water and nitrogen via contact electrification.

We synthesized ammonia (NH3) by bubbling nitrogen (N2) gas into bulk liquid water (200 mL) containing 50 mg polytetrafluoroethylene (PTFE) particles (~5 µm in diameter) suspended with the help of a surfactant (Tween 20, ~0.05 vol.%) at room temperature (25 °C). Electron spin resonance spectroscopy and density functional theory calculations reveal that water acts as the proton donor for the reduction of N2. Moreover, isotopic labeling of the N2 gas shows that it is the source of nitrogen in the ammonia. We propose a mechanism for ammonia generation based on the activation of N2 caused by electron transfer and reduction processes driven by contact electrification. We optimized the pH of the PTFE suspension at 6.5 to 7.0 and employed ultrasonic mixing. We found an ammonia production rate of ~420 µmol L-1 h-1 per gram of PTFE particles for the conditions described above. This rate did not change more than 10% over an 8-h period of sustained reaction.

Liquid-liquid transition and ice crystallization in a machine-learned coarse-grained water model.

Mounting experimental evidence supports the existence of a liquid-liquid transition (LLT) in high-pressure supercooled water. However, fast crystallization of supercooled water has impeded identification of the LLT line TLL(p) in experiments. While the most accurate all-atom (AA) water models display a LLT, their computational cost limits investigations of its interplay with ice formation. Coarse-grained (CG) models provide over 100-fold computational efficiency gain over AA models, enabling the study of water crystallization, but have not yet shown to have a LLT. Here, we demonstrate that the CG machine-learned water model Machine-Learned Bond-Order Potential (ML-BOP) has a LLT that ends in a critical point at pc = 170 ± 10 MPa and Tc = 181 ± 3 K. The TLL(p) of ML-BOP is almost identical to the one of TIP4P/2005, adding to the similarity in the equation of state of liquid water in both models. Cooling simulations reveal that ice crystallization is fastest at the LLT and its supercritical continuation of maximum heat capacity, supporting a mechanistic relationship between the structural transformation of water to a low-density liquid (LDL) and ice formation. We find no signature of liquid-liquid criticality in the ice crystallization temperatures. ML-BOP replicates the competition between formation of LDL and ice observed in ultrafast experiments of decompression of the high-density liquid (HDL) into the region of stability of LDL. The simulations reveal that crystallization occurs prior to the coarsening of the HDL and LDL domains, obscuring the distinction between the highly metastable first-order LLT and pronounced structural fluctuations along its supercritical continuation.

Terahertz radiation induced by shift currents in liquids.

Considerable progress has been made in the experimental studies on laser-induced terahertz (THz) radiation in liquids. Liquid THz demonstrates many unique features different from the gas and plasma THz. For example, the liquid THz can be efficiently produced by a monochromatic laser. Its yield is maximized with a longer driving-pulse duration. It is also linearly dependent on the excitation pulse energy. In two-color laser fields, an unexpected unmodulated THz field was measured, and its energy dependence of the driving laser is completely different from that of the modulated THz waves. However, the underlying microscopic mechanism is still unclear due to the difficulties in the description of ultrafast dynamics in complex disordered liquids. Here we propose a shift-current model. The experimental observations could be reproduced by our theory successfully. In addition, our theory could be further utilized to investigate the nuclear quantum effect in the THz radiation in H2O and D2O. This work provides fundamental insights into the origin of the THz radiation in bulk liquids.

Null mutants of a tomato Rho of plants exhibit enhanced water use efficiency without a penalty to yield.

Improving water use efficiency in crops is a significant challenge as it involves balancing water transpiration and CO2 uptake through stomatal pores. This study investigates the role of SlROP9, a tomato Rho of Plants protein, in guard cells and its impact on plant transpiration. The results reveal that SlROP9 null mutants exhibit reduced stomatal conductance while photosynthetic CO2 assimilation remains largely unaffected. Notably, there is a notable decrease in whole-plant transpiration in the rop9 mutants compared to the wild type, especially during noon hours when the water pressure deficit is high. The elevated stomatal closure observed in rop9 mutants is linked to an increase in reactive oxygen species formation. This is very likely dependent on the respiratory burst oxidase homolog (RBOH) NADPH oxidase and is not influenced by abscisic acid (ABA). Consistently, activated ROP9 can interact with RBOHB in both yeast and plants. In diverse tomato accessions, drought stress represses ROP9 expression, and in Arabidopsis stomatal guard cells, ABA suppresses ROP signaling. Therefore, the phenotype of the rop9 mutants may arise from a disruption in ROP9-regulated RBOH activity. Remarkably, large-scale field experiments demonstrate that the rop9 mutants display improved water use efficiency without compromising fruit yield. These findings provide insights into the role of ROPs in guard cells and their potential as targets for enhancing water use efficiency in crops.

Stabilizing Ti3C2Tx MXene flakes in air by removing confined water.

MXenes have demonstrated potential for various applications owing to their tunable surface chemistry and metallic conductivity. However, high temperatures can accelerate MXene film oxidation in air. Understanding the mechanisms of MXene oxidation at elevated temperatures, which is still limited, is critical in improving their thermal stability for high-temperature applications. Here, we demonstrate that Ti[Formula: see text]C[Formula: see text]T[Formula: see text] MXene monoflakes have exceptional thermal stability at temperatures up to 600[Formula: see text]C in air, while multiflakes readily oxidize in air at 300[Formula: see text]C. Density functional theory calculations indicate that confined water between Ti[Formula: see text]C[Formula: see text]T[Formula: see text] flakes has higher removal energy than surface water and can thus persist to higher temperatures, leading to oxidation. We demonstrate that the amount of confined water correlates with the degree of oxidation in stacked flakes. Confined water can be fully removed by vacuum annealing Ti[Formula: see text]C[Formula: see text]T[Formula: see text] films at 600[Formula: see text]C, resulting in substantial stability improvement in multiflake films (can withstand 600[Formula: see text]C in air). These findings provide fundamental insights into the kinetics of confined water and its role in Ti[Formula: see text]C[Formula: see text]T[Formula: see text] oxidation. This work enables the use of stable monoflake MXenes in high-temperature applications and provides guidelines for proper vacuum annealing of multiflake films to enhance their stability.

Concurrent warming and browning eliminate cold-water fish habitat in many temperate lakes.

Cold-water species in temperate lakes face two simultaneous climate-driven ecosystem changes: warming and browning of their waters. Browning refers to reduced transparency arising from increased dissolved organic carbon (DOC), which absorbs solar energy near the surface. It is unclear whether the net effect is mitigation or amplification of climate warming impacts on suitable oxythermal habitat (<20 °C, >5 mgO/L) for cold-loving species because browning expands the vertical distribution of both cool water and oxygen depletion. We analyzed long-term trends and high-frequency sensor data from browning lakes in New York's Adirondack region to assess the contemporary status of summertime habitat for lacustrine brook trout. Across two decades, surface temperatures increased twice as fast and bottom dissolved oxygen declined >180% faster than average trends for temperate lakes. We identify four lake categories based on oxythermal habitat metrics: constrained, squeezed, overheated, and buffered. In most of our study lakes, trout face either seasonal loss (7 of 15) or dramatic restriction (12 to 21% of the water column; 5 of 15) of suitable habitat. These sobering statistics reflect rapid upward expansion of oxygen depletion in lakes with moderate or high DOC relative to compression of heat penetration. Only in very clear lakes has browning potentially mitigated climate warming. Applying our findings to extensive survey data suggests that decades of browning have reduced oxythermal refugia in most Adirondack lakes. We conclude that joint warming and browning may preclude self-sustaining cold-water fisheries in many temperate lakes; hence, oxythermal categorization is essential to guide triage strategies and management interventions.

Chelation-directed interface engineering of in-place self-cleaning membranes.

Water-energy sustainability will depend upon the rapid development of advanced pressure-driven separation membranes. Although energy-efficient, water-treatment membranes are constrained by ubiquitous fouling, which may be alleviated by engineering self-cleaning membrane interfaces. In this study, a metal-polyphenol network was designed to direct the armorization of catalytic nanofilms (ca. 18 nm) on inert polymeric membranes. The chelation-directed mineralized coating exhibits high polarity, superhydrophilicity, and ultralow adhesion to crude oil, enabling cyclable crude oil-in-water emulsion separation. The in-place flux recovery rate exceeded 99.9%, alleviating the need for traditional ex situ cleaning. The chelation-directed nanoarmored membrane exhibited 48-fold and 6.8-fold figures of merit for in-place self-cleaning regeneration compared to the control membrane and simple hydraulic cleaning, respectively. Precursor interaction mechanisms were identified by density functional theory calculations. Chelation-directed armorization offers promise for sustainable applications in catalysis, biomedicine, environmental remediation, and beyond.

Evidence of ferroelectric features in low-density supercooled water from ab initio deep neural-network simulations.

Over the last decade, an increasing body of evidence has emerged, supporting the existence of a metastable liquid-liquid critical point in supercooled water whereby two distinct liquid phases of different densities coexist. Analyzing long molecular dynamics simulations performed using deep neural-network force fields trained to accurate quantum mechanical data, we demonstrate that the low-density liquid phase displays a strong propensity toward spontaneous polarization, as witnessed by large and long-lived collective dipole fluctuations. Our findings suggest that the dynamical stability of the low-density phase, and hence the transition from high-density to low-density liquid, is triggered by a collective process involving an accumulation of rotational angular jumps, which could ignite large dipole fluctuations. This dynamical transition involves subtle changes in the electronic polarizability of water molecules which affects their rotational mobility within the two phases. These findings hold the potential for catalyzing activity in the search for dielectric-based probes of the putative second critical point.