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Croton L. (Crotonoideae, Euphorbiaceae) is one of the largest angiosperm genera, comprising more than 1100 species with a pantropical distribution; however, the chloroplast (cp) genomes of a few species have been reported. In this study, the complete cp genome of Croton bonplandianus Baill. was newly sequenced, and the cp genome of Croton alabamensis E.A.Sm. ex Chapm. was assembled from publicly available data. These genomes were analyzed together with seven previously published Croton cp genomes to investigate structural variation, inverted repeat (IR) boundary dynamics, and phylogenetic relationships. The nine cp genomes exhibited substantial size variation (150,021–177,025 bp), driven primarily by pronounced differences in IR length (10,100–36,116 bp). Six distinct IR boundary configurations were identified, with IR expansion occurring predominantly at the expense of the large single‐copy (LSC) region rather than the small single‐copy (SSC) region. Phylogenomic analysis based on 78 shared protein‐coding genes supported the monophyly of nine Croton species and placed C. alabamensis and C. bonplandianus as successive early‐diverging lineages among the nine sampled species. Comparative analyses further identified 10 highly polymorphic cp genes as candidate molecular markers for phylogenetic and population‐level studies in the nine species examined, though their utility will require validation before application to additional species. Among the nine studied species, IR contraction and expansion in Croton were largely species‐specific and showed limited phylogenetic conservation. IR boundary shifts at cp genome junctions involved predominantly the LSC region, contrasting with the SSC‐driven patterns commonly reported in other angiosperm lineages; however, broader taxon sampling is needed to determine whether this reflects a genus‐wide pattern or is an artifact of limited sampling. Croton chloroplast genomes exhibit substantial structural diversity driven mainly by asymmetric inverted repeats (IRs) contraction and expansion, despite highly conserved gene content. IR boundary shifts predominantly involved the LSC region, indicating an LSC‐driven evolutionary pattern. The preliminary phylogenomic analyses confirmed the monophyly of Croton and resolved C. alabamensis and C. bonplandianus as early‐diverging lineages. The largely species‐specific IR configurations and the identification of highly polymorphic loci provide a valuable genomic foundation for future systematic, evolutionary, and conservation studies in Euphorbiaceae. However, these results are based on only nine species, and further sampling is needed to draw a final conclusion.

The Amino Acid–Polyamine-Organocation (APC) transporter GadC contributes to the survival of pathogenic bacteria under extreme acid stress by exchanging extracellular glutamate for intracellular γ-aminobutyric acid (GABA). Its structure, determined in an inward-facing conformation at alkaline pH, consists of the canonical LeuT-fold with a conserved five-helix inverted repeat, thereby resembling functionally divergent transporters such as the serotonin transporter SERT and the glucose-sodium symporter SGLT1. However, despite this structural similarity, it is unclear if the conformational dynamics of antiporters such as GadC follow the blueprint of these or other LeuT-fold transporters. Here, we used double electron-electron resonance (DEER) spectroscopy to monitor the conformational dynamics of GadC in lipid bilayers in response to acidification and substrate binding. To guide experimental design and facilitate the interpretation of the DEER data, we generated an ensemble of structural models in multiple conformations using a recently introduced modification of AlphaFold2 . Our experimental results reveal acid-induced conformational changes that dislodge the Cterminus from the permeation pathway coupled with rearrangement of helices that enables isomerization between inward- and outward-facing states. The substrate glutamate, but not GABA, modulates the dynamics of an extracellular thin gate without shifting the equilibrium between inward- and outward-facing conformations. In addition to introducing an integrated methodology for probing transporter conformational dynamics, the congruence of the DEER data with patterns of structural rearrangements deduced from ensembles of AlphaFold2 models illuminates the conformational cycle of GadC underpinning transport and exposes yet another example of the divergence between the dynamics of different families in the LeuT-fold.

While thermal rates of state transitions in classical systems have been studied for almost a century, associated transition-path times have only recently received attention. Uphill and downhill transition paths between states at different free energies should be statistically indistinguishable. Here, we systematically investigate transition-path-time symmetry and report evidence of its breakdown on the molecular- and meso-scale out of equilibrium. In automated Brownian dynamics experiments, we establish first-passage-time symmetries of colloids driven by femtoNewton forces in holographically-created optical landscapes confined within microchannels. Conversely, we show that transitions which couple in a path-dependent manner to fluctuating forces exhibit asymmetry. We reproduce this asymmetry in folding transitions of DNA-hairpins driven out of equilibrium and suggest a topological mechanism of symmetry breakdown. Our results are relevant to measurements that capture a single coordinate in a multidimensional free energy landscape, as encountered in electrophysiology and single-molecule fluorescence experiments. Microscopic transition mechanisms impact many biophysical systems. In this work, the authors explore transition path times between thermodynamic states experimentally, and show symmetry breaking in the transition times under an external force that drives the system out of equilibrium.

Much of life on Earth, including plants, fungi, and bacteria, evolved to resist toxic environmental fluoride. In eukaryotes, the major resistance mechanism is fluoride export by membrane proteins known as FEX. Using electrophysiology and transport assays, we establish that FEX from plants and yeasts are highly selective fluoride channels. Fluoride transport activity depends on reversible sodium ion binding, but sodium itself is not transported. We determine a structure of a FEX protein, from pathogenic yeast Candida albicans , using cryo-EM. Bolstered by mutagenesis studies, this structure reveals a fluoride permeation route through a single phenylalanine-lined pore. Molecular dynamics simulations demonstrate that a cation binding motif adjacent to the pore provides a stable sodium binding site that is accessible from the external aqueous solution. Comparison to the structurally related bacterial fluoride channels, Flucs, provides a glimpse of the evolution of structural and mechanistic complexity in a membrane protein family with inverted repeat architecture. Eukaryotes, such as fungi, protozoa, and plants, resist fluoride using fluoride exporters called FEX. Here, authors determine a molecular structure of FEX from Candida albicans, and show that FEX is a single-pore, sodium-activated fluoride channel.

The assembly of nanoparticles into three-dimensional (3D) architectures could allow for greater control of the interactions between these particles or with molecules. DNA tubes are known to form through either self-association of multi-helix DNA bundle structures or closing up of 2D DNA tile lattices. By the attachment of single-stranded DNA to gold nanoparticles, nanotubes of various 3D architectures can form, ranging in shape from stacked rings to single spirals, double spirals, and nested spirals. The nanoparticles are active elements that control the preference for specific tube conformations through size-dependent steric repulsion effects. For example, we can control the tube assembly to favor stacked-ring structures using 10-nanometer gold nanoparticles. Electron tomography revealed a left-handed chirality in the spiral tubes, double-wall tube features, and conformational transitions between tubes.

Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1's proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102-104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18-21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.

Background Raw second-generation (2G) lignocellulosic biomass materials have the potential for development into a sustainable and renewable source of energy. Poplar is regarded as a promising 2G material (P. davidiana Dode×P. bolleana Lauch, P. bolleana, P. davidiana, P. euphratica, et al). However, their large-scale commercialization still faces many obstacles. For example, drought prevents sufficient irrigation or rainfall, which can reduce soil moisture and eventually destroy the chloroplast, the plant photosynthetic organelle. Heterosis is widely used in the production of drought-tolerant materials, such as the superior clone “Shanxinyang” selected from the offspring of Populus davidiana Dode×Populus bolleana Lauch. Because it produces good wood and is easily genetically transformed, “Shanxinyang” has become a promising material for use in tree genetics. It is also one of the most abundant biofuel plants in northern China. Understanding the genetic features of chloroplasts, the cp transcriptome and physiology is crucial to elucidating the chloroplast drought-response model. Results In this study, the whole genome of “Shanxinyang” was sequenced. The chloroplast genome was assembled, and chloroplast structure was analysed and compared with that of other popular plants. Chloroplast transcriptome analysis was performed under drought conditions. The total length of the “Shanxinyang” chloroplast genome was 156,190 bp, the GC content was 36.75%, and the genome was composed of four typical areas (LSC, IRa, IRb, and SSC). A total of 114 simple repeats were detected in the chloroplast genome of “Shanxinyang”. In cp transcriptome analysis, we found 161 up-regulated and 157 down-regulated genes under drought, and 9 cpDEGs was randomly selected to conduct reverse transcription (RT)–qPCR., in which the Log2 (fold change) was significantly consistent with the qPCR results. The analysis of chloroplast transcription under drought provided clues for understanding chloroplast function under drought. The phylogenetic position of “Shanxinyang” within Populus was analysed by using the chloroplast genome sequences of 23 Populus plants, showing that “Shanxinyang” belongs to Sect. Populus and is sister to Populus davidiana. Further, mVISTA analysis showed that the variation in non-coding (regulatory) regions was greater than that in coding regions, which suggests that further attention should be paid to the chloroplast in order to obtain new evolutionary or functional insights related to aspects of plant biology. Conclusions Our findings indicate that complex prokaryotic genome regulation occurs when processing transcripts under drought stress. The results not only offer clues for understanding the chloroplast genome and transcription features in woody plants but also serve as a basis for future molecular studies on poplar species.

Background The Synurophyceae is one of most important photosynthetic stramenopile algal lineages in freshwater ecosystems. They are characterized by siliceous scales covering the cell or colony surface and possess plastids of red-algal secondary or tertiary endosymbiotic origin. Despite their ecological and evolutionary significance, the relationships amongst extant Synurophyceae are unclear, as is their relationship to most other stramenopiles. Results Here we report a comparative analysis of plastid genomes sequenced from five representative synurophycean algae. Most of these plastid genomes are highly conserved with respect to genome structure and coding capacity, with the exception of gene re-arrangements and partial duplications at the boundary of the inverted repeat and single-copy regions. Several lineage-specific gene loss/gain events and intron insertions were detected (e.g., cem A, dna B, syf B, and trn L). Conclusions Unexpectedly, the cem A gene of Synurophyceae shows a strong relationship with sequences from members of the green-algal lineage, suggesting the occurrence of a lateral gene transfer event. Using a molecular clock approach based on silica fossil record data, we infer the timing of genome re-arrangement and gene gain/loss events in the plastid genomes of Synurophyceae.

Transition paths represent the parts of a reaction where the energy barrier separating products and reactants is crossed. They are essential to understanding reaction mechanisms, yet many of their properties remain unstudied. Here, we report measurements of the average shape of transition paths, studying the folding of DNA hairpins as a model system for folding reactions. Individual transition paths were detected in the folding trajectories of hairpins with different sequences held under tension in optical tweezers, and path shapes were computed by averaging all transitions in the time domain, 〈t(x)〉, or by averaging transitions of a given duration in the extension domain, 〈x(t|τ)〉 τ . Whereas 〈t(x)〉 was close to straight, with only a subtle curvature, 〈x(t|τ)〉 τ had more pronounced curvature that fit well to theoretical expectations for the dominant transition path, returning diffusion coefficients similar to values obtained previously from independent methods. Simulations suggested that 〈t(x)〉 provided a less reliable representation of the path shape than 〈x(t|τ)〉 τ , because it was far more sensitive to the effects of coupling the molecule to the experimental force probe. Intriguingly, the path shape variance was larger for some hairpins than others, indicating sequence-dependent changes in the diversity of transition paths reflective of differences in the character of the energy barriers, such as the width of the barrier saddle-point or the presence of parallel paths through multiple barriers between the folded and unfolded states. These studies of average path shapes point the way forward for probing the rich information contained in path shape fluctuations.

Sodium-dependent transporters couple the flow of Na⁺ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na⁺-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na⁺ and ligand influence the equilibrium between other states remains unknown. Here, double electron–electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na⁺, vSGLT favors an inward-facing conformation. Upon binding both Na⁺ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na⁺ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.