Proceedings of the National Academy of Sciences

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is foundational to life on Earth, catalyzing carbon dioxide (CO2) fixation to generate biomass. However, Rubisco is a slow and inefficient enzyme that has proven challenging to engineer. We applied the structure-informed machine learning (ML) model ESM-IF1 to identify plausible amino acid sites in the large subunit of Nicotiana tabacum Rubisco to target for directed evolution. ML-assisted library design followed by selection in Rubisco-dependent Escherichia coli identified multiple enriched variants displaying improved catalytic efficiency. Several improved variants carried amino acid changes not found in the evolutionary lineage of plants, despite being assembly competent in plant chloroplasts, demonstrating that ML-assisted protein design can explore functional sequence space beyond what is observed from natural sequence diversity. Most prominently, the T391I substitution improved carboxylation rate by 29% and aerobic carboxylation efficiency by 43%. Our findings demonstrate the utility of ML-assisted evolution for engineering Rubisco with improved carboxylation efficiency and potential for enhancing crop productivity.

Nearly all carbon on Earth today is fixed by the enzyme ribulose-1,5-bisphopshate carboxylase/oxygenase ( rubisco), which converts carbon dioxide (CO2) to sugar phosphates. All rubiscos measured thus far display a kinetic isotope effect (KIE) where 12CO2 is fixed at a faster rate than 13CO2. The relationship between rubiscos KIE and the carbon isotope composition of plants, algae, and organic matter is central to many fields in the Earth sciences, plant biology, and biochemistry. Currently, all applications assume that the KIE does not vary with temperature. Here, we examine this assumption experimentally with in vitro KIE measurements of two rubiscos from phylogenetically distinct host organisms and rubisco protein clades - a Form I rubisco from the plant, Spinacia oleracea (spinach) and a Form II rubisco from the bacterium Rhodosprillium rubrum. We that find that both KIEs decrease linearly by [~]4.5{per thousand} from 10-35{degrees}C with statistically indistinguishable slopes. We place these results into biological and geologic contexts by comparing them to observed variations in the carbon isotope composition of modern terrestrial plants and marine organic carbon, the geologic carbon isotope record, and rubiscos biochemistry. We show that the measured temperature dependencies are sufficiently large to impact our interpretations of the enzymatic processes that drive variations in rubisco KIEs, as well as applications of stable carbon isotopes in the Earth and biological sciences. Significance StatementThe carbon isotope composition of plants, algae, and organic matter are interpreted with models that assume the kinetic isotope effect of the carbon-fixing enzyme rubisco is temperature-independent, even though temperature varies by tens of degrees across the Earth today and in the past. Here, we demonstrate that the kinetic isotope effect of rubisco is temperature-dependent, suggesting that some of this isotopic variation may be due to intrinsic enzyme properties alone. In addition, though the rubiscos we measured are from diverse organisms (plant vs. bacteria), their KIEs show statistically indistinguishable temperature dependencies. This data forms the basis for future thermodynamic models on rubisco biochemistry.

Evolutionary innovation is a key driver of the colonization of new environments and the adaptive radiations of major groups. Novel traits typically evolve through the modification of pre-existing characters but the genetic paths underlying their origin have been challenging to trace, and the general requirements for and relative order of different kinds of gene mutations have been difficult to assess. Here, we trace the genomic origins of four procoagulant venom toxins (factor X, factor V, group I phospholipase A2, and Kunitz-type toxins) that collectively underlie a novel, especially potent blood-clotting venom type in the recently evolved Australian brown snake and taipan clade. We discover evidence for a previously unknown fifth toxin, coagulation factor VII, and show that the toxins evolved through two distinct genetic paths. The factor X and factor V toxins evolved through the sequential de novo co-option of ancestral clotting factor proteins that entailed their heterotopic expression in the venom gland, the fixation of segmental duplications containing each locus, and subsequent gain-of-function mutations that rendered factor X and factor V constitutively active. In contrast, the phospholipase A2 and Kunitz-type toxins evolved by modifying the functions of neurotoxins that were part of the venom arsenal. Our findings support models in which innovative mutations in single-copy genes precede gene duplication in the evolution of novel proteins and offer a rare view into the genesis of a complex trait that has played a central role in a major adaptive radiation. Significance StatementThis study investigates how an entirely new blood-clotting venom type evolved during the recent radiation of Australias iconic venomous snakes. We traced the key genetic events that occurred on the evolutionary path to one of the worlds most potent venoms. We found that the novel venom activity evolved through the sequential co-option of multiple proteins of the snakes own blood-clotting system, followed by the modification of two venom neurotoxins into proteins with procoagulant activities. We suggest that these unique de novo gene co-options are seminal events that can unlock new ecological strategies, which in turn, may enable major adaptive radiations.

Monoterpenes (C10) are a large group of specialized metabolites important for plant interactions with the environment. Their biosynthesis is well understood in seed plants, where geranyl diphosphate serves as the canonical substrate, but knowledge of monoterpene biosynthesis outside seed plants remains very limited. Here, we report neryl diphosphate (NPP)-derived monoterpene biosynthesis via a biosynthetic gene cluster in the liverwort Marchantia polymorpha. MpMTPSL2, a microbial-type terpene synthase, converts NPP into -phellandrene and D-limonene in vitro. CRISPR knockout lines showed reduced production of both monoterpenes, providing direct genetic evidence for its in planta function. MpCPT5, a cis-prenyltransferase (CPT) family member identified through co-expression with MpMTPSL2, was confirmed to encode NPP synthase, as its knockout plants abolished -phellandrene and D-limonene production. Subcellular localization analyses in protoplasts and stable transgenic plants demonstrated that both MpCPT5 and MpMTPSL2 localize to plastids, co-localizing across all cell types with markedly stronger signals in non-green plastids of oil-body cells. Consistent with this, expression of both genes under their respective promoters was nearly abolished in oil-body-deficient mutants and strongly upregulated in a gain-of-function line for oil-body formation. MpMTPSL2 and MpCPT5 are physically linked through a shared bidirectional promoter that drives their coexpression specific to oil body cells, forming a unique biosynthetic gene cluster whose coordinated expression is maintained by PRC2-mediated H3K27me3 repression. Phylogenetic analysis implies that NPP synthases in M. polymorpha and in flowering plants evolved independently from their respective long-chain CPT ancestors. These findings provide new insights into the mechanisms and evolution of monoterpene biosynthesis in non-seed plants. Significance statementMonoterpenes are a diverse group of specialized metabolites produced widely among land plants, yet our understanding of their biosynthesis outside seed plants remains limited. Here we report that in the liverwort Marchantia polymorpha, the non-canonical substrate neryl diphosphate is used for monoterpene biosynthesis. The functions of the monoterpene synthase gene MpMTPSL2 and the neryl diphosphate synthase gene MpCPT5 were demonstrated through CRISPR knockouts. These two genes are physically linked and share a bidirectional promoter. Promoter assays show both genes function in plastids within oil-body cells, revealing cell-type specificity. These findings shew new light on the mechanisms and evolution of monoterpene biosynthesis in non-seed plants.

Circadian clocks generate stable [~]24-h rhythms with a defined period, temperature compensation, and entrainment to external cues that set phase. However, the molecular reactions that generate these features are not fully understood. In cyanobacteria, timekeeping is driven by the hexameric ATPase KaiC, which consists of two homologous domains, CI and CII, and whose enzymatic turnover underlies rhythmic phosphorylation. Here we identify ADP release as the rate-limiting step in the KaiC ATPase cycle and demonstrate that the KaiC ATPase nucleotide cycle integrates core properties of the circadian clock. Across KaiC period mutants, increased ADP occupancy is associated with longer periods. Elevated temperature shifts KaiC toward an ADP-bound state, offsetting the thermal acceleration of ATP hydrolysis. KaiB reinforces this ADP-bound conformation by inhibiting nucleotide exchange, thereby strengthening inhibition of CI ATPase activity of KaiC and tuning oscillation amplitude in a temperature-dependent manner. In contrast, KaiA accelerates ADP-to-ATP exchange within the KaiB-KaiC complex, thereby stimulating CI ATPase activity and promoting KaiB-KaiC dissociation prior to KaiC phosphorylation. Phosphorylation begins only after this transition, indicating that KaiC nucleotide-bound state sets the phase of the phosphorylation cycle. Collectively, these results establish the nucleotide cycle of KaiC ATPase as a unifying mechanism that connects molecular reactions to the defining properties of the cyanobacterial circadian clock.

The evolutionary emergence of complex organs requires the integration of distinct developmental modules, yet the mechanisms bridging divergent tissue programs into unified functional units remain poorly understood. Animal venom systems, combining high-output secretory epithelia with muscular delivery systems, a provide an exceptional model for studying this modular integration. Here, we show that the venom gland of the spider Parasteatoda tepidariorum is a developmental chimera built by the hierarchical co-option of three ancestral programs. The appendage-patterning gene Distal-less establishes the primary spatial coordinate system for gland development. Downstream, the transcription factor sage maintains secretory epithelium identity; its loss causes downregulation of key venom-processing enzymes and triggers a dramatic phenotypic reversion to a neuronal state. This neural upregulation, accompanied by the downregulation of Notch ligands (delta and jagged), reveals that sage actively represses a latent neural program to maintain secretory fate. Crucially, the myogenic factor sum-1 governs the surrounding muscle layer to drive paracrine crosstalk (Wnt) and sustain lipid metabolism. Knockdown of sum-1 downregulates wntless and fatty acid anabolism genes, alongside the abnormal accumulation of lipid droplets in the secretory epithelium. Ultimately, our findings demonstrate how a complex organ is assembled by fusing an appendicular address, a repressed neuroglandular program, and a myogenic metabolic driver. This modular architecture illustrates a fundamental principle in evolutionary developmental biology: novelty can arise by harnessing the physiological and metabolic capacities of adjacent tissues to fuel the physiological demands of a newly emerged organ. Significance statementHow novel organs evolve by integrating distinct developmental modules remains a fundamental question in biology. Using the spider venom gland as a model, we show that this complex organ is a developmental chimera built by recruiting three ancestral tissue programs. The appendage-selector gene Distal-less (Dll) establishes the glands primary proximal-distal structural axis. Downstream, the transcription factor sage maintains secretory epithelial identity by actively repressing a default neural program. Concurrently, the myogenic regulator sum-1 sustains epithelial lipid metabolism through paracrine Wnt signaling. These findings establish a mosaic model of organogenesis, demonstrating that radical evolutionary innovation can arise by forging morphogenetic and metabolic partnerships that harness adjacent tissues to fuel newly emerged structures.

Biophysical pleiotropy, the phenomenon in which a mutation affects multiple protein properties, underlies many genetic diseases and shapes protein evolution, yet remains poorly understood, limiting synthetic biology and therapeutic development. Although extensively studied in soluble proteins, little is known about how pleiotropic effects shaped the evolution of receptor protein functions. Here, we developed a cell-based assay designed to assess pleiotropic effects in G protein-coupled receptors (GPCRs), the largest receptor class in eukaryotes. Because our assay design allows for multiple GPCR molecular phenotypes (basal activity, ligand-dependent signaling, and cellular receptor abundance) to be simultaneously assessed, we applied it to study rhodopsin, a visual GPCR that evolved high light sensitivity by maximizing light-driven responses while minimizing thermally driven noise. To investigate this, we integrated our assay with deep mutational scanning of relevant rhodopsin domains to test the impacts of [~]2,000 single-residue substitutions in rhodopsin across all three molecular phenotypes ([~]6,000 measurements). By analyzing these data in the context of rhodopsins protein structures, we discovered complex pleiotropic effects that act asymmetrically to impose joint constraints, restricting mutational tolerance in rhodopsins retinal binding pocket and G protein interaction interface. A dose-response analysis of rhodopsin signaling revealed that these pleiotropic constraints arise from the dual functions of its chromophore, which acts as an agonist upon light-driven isomerization but also an inverse agonist in darkness. Because these constraints reflect mechanistically driven limitations in the evolution of receptor signaling, these findings reveal a role for biophysical pleiotropy in shaping the sensory capabilities of receptor proteins. Significance StatementUnderstanding how genetic variation impacts protein function is an important but challenging area of research as many mutations are pleiotropic, simultaneously affecting multiple protein properties, including structure, stability, and activity. Previously, genetic screens have systematically mapped pleiotropic effects in soluble proteins, but transmembrane receptor proteins embedded in cellular membranes are much more difficult to assay. These are key receptors that convert environmental stimuli into appropriate physiological responses. Here, we present a systematic study of pleiotropic effects within the receptor responsible for dim-light vision in vertebrates, revealing how light sensitivity can be mediated through differential conformational states of its chromophore. This is important not only for understanding how receptors evolved enhanced capabilities, but also for the development of tunable protein systems.

Cyanobacteriochromes (CBCRs) are bilin-binding photoreceptors with remarkable spectral versatility. Using phycocyanobilin (PCB) as a chromophore, CBCRs regulate diverse light-dependent processes in cyanobacteria, ranging from photosynthesis to chromatic acclimation. Although extensive studies have uncovered multiple spectral tuning mechanisms in bilin-binding proteins, recent structural studies of far-red CBCRs suggest the existence of additional tuning strategies in both the 15Z and 15E states. Here we report crystal structures of the representative far-red CBCR Anacy_2551g3 in three distinct light-absorbing states, all of which adopt a compact all-syn PCB conformation. These structures demonstrate that 15Z/15E photoisomerization in Anacy_2551g3 involves minimal chromophore rotation relative to the GAF domain, in stark contrast to other characterized bilin-based photoreceptors. To investigate the molecular basis of its far-red absorption, we examined the protonation and tautomeric states of PCB in the Pfr state using resonance Raman (RR) spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Comparisons of experimental and calculated RR spectra support a bilin lactam as the predominant tautomeric form in the Pfr state. Integrating structural, spectroscopic, computational and mutational analyses, we propose that specific protein-chromophore interactions play critical roles in modulating chromophore conjugation beyond bilin coplanarity. Structural analyses further suggest a signaling model in which light regulation by Anacy_2551g3 is mediated through reversible switching between a high-affinity Pfr state and a low-affinity Po state that does not involve large chromophore motions. Together, these results provide new insights into how protein-chromophore coupling governs spectral tuning and light signaling in bilin-based photoreceptors. Significance statementBilins are widespread biological pigments that mediate photoreception, light harvesting, and photosynthesis across diverse light environments. In a phenomenon known as spectral tuning, the optical properties of bilin-binding proteins are profoundly influenced by protein-chromophore interactions. Mechanistic understanding of spectral tuning and light signaling is important not only for advancing fundamental knowledge of light-sensitive proteins but also for developing new engineering strategies in synthetic biology and biotechnology. Recently discovered cyanobacteriochromes (CBCRs) exhibit remarkable spectral diversity and structural versatility, providing excellent model systems for dissecting the mechanisms of bilin-based photoreceptors. By integrating crystallography, spectroscopy and computational methods, this work examines three distinct light absorbing states of a representative far-red CBCR. Our findings reveal previously unrecognized mechanisms of spectral tuning and light signaling, highlighting the critical roles of protein-chromophore coupling and electrostatic interactions in regulating photoreceptor function.

Strains of the cyanobacterium Acaryochloris marina exhibit diverse far-red light-harvesting properties during chlorophyll d-based photosynthesis. Here, we show that differences in light absorption among A. marina strains arise exclusively from Photosystem I (PSI) and reflect variation in multiple low-energy chlorophyll states. Time-resolved fluorescence reveals different combinations of low-energy states among strains, generating a continuum of spectral phenotypes. Cryo-EM structures of PSI at [~]1.8 [A] resolution reveal similar low-energy states arising from distinct pigment environments, demonstrating that red-shifted absorption is not governed by a single conserved motif. Phylogenetic analyses show that spectral tuning evolved through modular variation and reassortment of PSI components. These results indicate that distinct pigment configurations can converge on similar low-energy states, extending light harvesting near the energetic limit of oxygenic photosynthesis.

Chlorosomes are the largest known photosynthetic light-harvesting antennas, yet unlike protein-based antennas, they lack protein scaffolds that organize pigment molecules and instead contain self-assembled tubular and lamellar bacteriochlorophyll aggregates. How these antennas achieve directional and efficient energy transfer has remained unresolved. By applying ultrafast transient absorption spectroscopy to individual wild-type and mutant chlorosomes, we resolved six kinetic components obscured by ensemble averaging and assigned each to either lamellar or tubular aggregates. Lamellar aggregates expand light-harvesting capacity, whereas tubular aggregates serve as the primary energy donors to the baseplate. Such structural heterogeneity is therefore not merely suppressed but tuned to balance light-harvesting capacity with robust energy delivery. These findings reveal a division-of-labor strategy among pigment aggregates for efficient light harvesting without protein scaffolds.

Protein kinase activation is driven by conformational changes across multiple structural components, including the conserved Asp-Phe-Gly (DFG) motif, but whether these transitions follow a universal mechanism remains unclear. Here we combine over 8.3 milliseconds of distributed unbiased molecular dynamics simulations with Markov state models (MSMs) to compare the conformational landscapes of the ABL1, EGFR and MET kinase domains. To maximize unbiased sampling of functionally relevant conformational space, we use a transfer seeding strategy that steers AlphaFold2 models derived from homologous templates to sample MET conformational states absent from available experimental databases. We find that related DFG-motif geometries separate into distinct kinetic networks. These shared structural states are connected by kinase-specific activation pathways with different regulatory elements controlling the slowest step of activation. Our findings reveal that the shared nomenclature masks distinct transition mechanisms between kinase domains, revealing new regions critical for activity and targetable conformations for inhibitor design.

Mutualisms play critical roles in organismal stress tolerance; yet environmental stressors may simultaneously alter the evolution of the mutualism itself. Stress may have particularly strong impacts on the evolution of microbial mutualists due to their capacity for rapid genetic change. Here we used a legume-rhizobia mutualism, in which plants exchange carbon for symbiotically fixed nitrogen, to evaluate how mutualisms evolve in response to stressors. We experimentally evolved populations of Rhizobium leguminasarum in a full factorial design, manipulating drought and nitrogen. We quantified genomic changes in Rhizobium populations as well as their quality as partners with their plant host, Trifolium repens. Drought selected for context- dependent stress benefits to the host; drought-adapted Rhizobium strains provided increased benefits to the host under drought, but fewer benefits to the host in well-watered environments. Conversely, nitrogen fertilization selected for decreased Rhizobium partner quality. Comparative genomics indicated that selection on standing structural variants along the symbiotic plasmid may underpin these drought benefits, specifically along genes associated with desiccation tolerance. These results suggest that stress can expand the benefits rhizobia provide to their legume hosts beyond nitrogen fixation, with rapid symbiont evolution an engine in promoting adaptive plant phenotypes. SIGNIFICANCE STATEMENTWhile mutualisms are fundamental to ecosystem functioning, they are often assumed to break down under environmental stress. We showed the opposite can happen. Here, we factorially manipulated drought stress and nitrogen fertilization and experimentally evolved a model legume-rhizobia mutualism, where rhizobial-bacteria trade nitrogen for plant carbon. When droughted, the symbiotic bacteria rapidly evolved new traits that made them better partners under drought, improving plant growth within just a few generations. Evolution occurred mainly on a specialized bacterial plasmid, a mobile piece of DNA distinct from the main genome. By contrast, added nitrogen fertilizer led to decreases in bacterial benefits. These findings suggest that mutualisms are not simply fragile in the face of global change, but can expand mutualistic benefits to buffer partners against stress. CLASSIFICATIONBiological Sciences, Evolution

Complex signaling pathways organize cell expansion and proliferation across cells to pattern tissues and organs in plants. The sulfotyrosine peptide hormone family, PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY), contributes to these processes. We identified two plasma membrane-localized receptors, PSYR1 and PSYR2, that are necessary for PSY signaling and regulate growth in Physcomitrium patens. Membrane-associated PSYRs accumulate to high levels in a mutant lacking TYROSYL PROTEIN SULFOTRANSFERASE (TPST). Given that a tpst null mutant ({Delta}tpst) is impaired in sulfation, this suggests that in the absence of sulfated peptides, PSYRs accumulate on the membrane. A null mutant of the PSY receptors, {Delta}psyr1/2, showed increased growth and was epistatic to {Delta}tpst, suppressing defects in gametophore formation and early senescence. The transcriptional profiles comparing wild type to {Delta}psyr1/2 and {Delta}psyr1/2/{Delta}tpst showed 25 to 30 differentially expressed genes between the receptor null mutants and wild type, with a common signature of cell wall remodeling and stress responses. Similarly, a PSYR1 kinase-inactive mutation rescued {Delta}tpst and relieved the accumulation of membrane-associated PSYRs. In contrast, overexpression of PSYRs inhibited plant growth, with phenotypic severity correlating with the amount of overexpression. These data are consistent with a constitutive activation model in which membrane-associated PSYRs unbound to PSY serve to inhibit growth through an active kinase. In the presence of the PSY peptide, the kinase is inactivated, promoting growth and driving PSY expression. The relationship between growth-repressive PSYR kinase activity and growth-promoting PSYR kinase inactivation in P. patens serves as a model for optimizing plant growth and development.

It is an open question whether variation in the genetic load of unconditionally deleterious mutations contributes substantially to the variability in human disease. Here, we solve for mutation-selection-drift balance and predict variation in genetic load given a realistic human genome-wide deleterious mutation rate, U, and a distribution of fitness effects (DFE). Empirical estimates of U come from sequence constraint, which fails to count slightly deleterious mutations that nevertheless fix. We use the inferred DFE to correct for this and conclude that total human U>3.8. Two humans typically differ in ancestral fitness by 17-33% given uncertainty in U, or by 6-49% when we consider a broad range of alternative DFEs. Results are similar for other species with larger mean selection coefficients, such as other mammals. Most variation in load comes from rare variants with frequencies below 1%, with a substantial fraction coming from ultra-rare variants below 0.01%. This could help explain why some of the heritability observed in pedigree studies is missing from genome-wide association studies. Accounting for rare and ultra-rare variants, e.g., via variant-effect prediction of unique mutations from whole-genome sequencing rather than via polygenic risk scores, could help identify individuals at high risk of disease. SignificanceMany human mutations mildly disrupt molecular function, e.g., by destabilizing proteins. Having too many of these mutations would have reduced fitness in ancestral human environments and might contribute to disease today. Here, we mathematically derive how much variation in fitness such mutations cause, using estimated human parameter values. Rare variants with larger fitness effects contribute the most. Identifying individuals with high disease risk likely requires methods capable of scoring rare variants.

Plasmodesmata are intercellular channels that mediate symplastic communication between plant cells. Molecular transport through these channels is critically regulated by dynamic callose deposition and degradation, yet the secretory mechanisms that deliver regulatory components to plasmodesmata remain poorly understood. Here, we identify and characterize a non-canonical plasmodesmata-associated module of the exocyst, an evolutionarily conserved protein complex involved in secretory vesicle tethering and exocytosis. Exocyst subunits EXO70G1, SEC15A, EXO84C, and SEC10A specifically accumulate at plasmodesmata, whereas the canonical exocyst subunits EXO70A1 and SEC8 do not. Genetic and interaction analyses show that EXO70G1 acts as a landmark for recruiting SEC15A and EXO84C to plasmodesmata, revealing a distinct mode of exocyst targeting at these membrane domains. EXO70G1-dependent exocyst targeting to plasmodesmata depends on phosphoinositides and sphingolipids, consistent with the specialized lipid environment of plasmodesmal membranes. Loss of EXO70G1 results in increased callose accumulation and reduced symplastic transport, and strongly enhances developmental defects of a callose-overproducing mutant. In addition, exo70G1 mutants display enhanced resistance to bacterial pathogen Pseudomonas syringae, linking reduced plasmodesmal permeability to anti-bacterial defense. Cross-species analysis further indicates that plasmodesmata association is a derived feature of the EXO70G clade, present in angiosperms but absent from non-angiosperm EXO70 homologs. Together, our findings show that exocyst diversification in plants has generated a specialized trafficking module - plasmodesmata-associated exocyst - that links vesicle delivery to callose homeostasis at plasmodesmata, thereby regulating intercellular communication, development, and immunity. TeaserA specialized secretion module of the exocyst complex regulates plant cell-to-cell connectivity by controlling callose turnover at plasmodesmata

Across kingdoms, cells fold their membranes into precise shapes closely linked to their functions. In mature land-plant chloroplasts, the photosynthetic membranes have been viewed as strictly lamellar and it is unknown whether they can take on a different structure while remaining functional. Here, we show that mature Arabidopsis thaliana chloroplasts can transform this network into a gyroid-type cubic membrane, which we call the gyrobody. The gyrobody forms reversibly during the night and preserves photosystem II photochemistry. A decrease in stromal side thylakoid surface charge, caused by lower protein phosphorylation, triggers the lamellar-to-gyroid transition which the curvature-inducing lipid MGDG facilitates. This shows that the mature plant thylakoid network is not locked into its lamellar form, revealing unexpected structural flexibility of this system.

Ocean warming and marine heatwaves threaten seagrasses, which are considered highly vulnerable to rising temperatures. We compared thermal stress responses of the tropical seagrass Halophila stipulacea collected from the Gulf of Aqaba in 2017 and 2022. Under mesocosm-applied thermal stress, 2017 plants showed reduced growth, impaired photochemistry, and extensive transcriptomic changes. In contrast, 2022 plants maintained normal growth and photochemistry with minimal transcriptional responses. The enhanced thermal stress tolerance of 2022 plants was associated with a "stress-ready" transcriptome, with many stress-response genes already up/downregulated under control conditions. These changes coincided with local ocean warming and a shift from episodic to chronic thermal stress. Our findings provide the first longitudinal dataset in a seagrass that tracks physiological and molecular changes occurring concurrently with documented ocean warming.

Fission is essential for proper mitochondrial function and for cellular homeostasis. Dysfunction in mitochondrial fission is associated with several neurological disorders, including the rare and lethal encephalopathy EMPF1, which is caused by de novo heterozygous DNM1L variants. DNM1L encodes the mitochondrial fission mechanoenzyme DRP1, which can intrinsically self-assemble and induce membrane scission. Wild-type DRP1 puncta that appear throughout the cytoplasm are thought to be pre-scission complexes of well-ordered oligomeric assemblies. Immunofluorescence imaging of patient-derived EMPF1 fibroblasts carrying assembly-deficient DNM1L variants reveals elongated mitochondrial networks consistent with impaired fission. Despite this loss-of-function phenotype, these cells retain essentially wild-type numbers of DRP1 puncta. We confirmed the previously reported inability of purified pathogenic DRP1 variants p.Gly363Asp and p.Gly401Ser to assemble under conditions in which WT DRP1 forms helical polymers. Under macromolecular crowding conditions, however, both wild-type and mutant DRP1 access condensed states whose formation depends on protein concentration and solution conditions. Acute treatment of EMPF1 fibroblasts with 1,6-hexanediol preferentially alters DRP1 puncta fluorescence intensity and distribution in mutant cells relative to wild type, indicating genotype-dependent differences in puncta material properties. Together, these findings support a model in which DRP1 puncta occupy a continuum of condensed states, only a subset of which mature into fission-competent assemblies, revealing biomolecular condensation as a previously unrecognized layer of DRP1 regulation. Biasing DRP1 along this continuum may provide a mechanistic basis for impaired fission in EMPF1 and suggest opportunities to restore productive assembly in select pathogenic contexts.

High pressure (HP) can trigger bacterial spore germination, acting either through germinant receptors (GRs) or the SpoVA channel. However, the mechanism by which HP activates these membrane-embedded proteins remains elusive. Here, using Bacillus subtilis, we demonstrate that the GerA germinant receptor (GR) is the primary target of moderate HP (50-300 MPa). Mutagenesis reveals that pore-lining residues within the GerA ion channel are essential for the pressure response, whereas canonical ligand-binding and intramembrane signaling residues are dispensable. We then propose a <underline>s</underline>tretch-<underline>t</underline>o-<underline>o</underline>pen (STO) model, in which HP differentially compresses the more compliant inner membrane (IM) relative to the rigid spore core, generating lateral membrane tension that promotes opening of the GerA channel. In situ membrane tension measurements indicate HP-induced compression of IM phospholipids and elevated membrane tension. This tension-dependent gating is further supported by the pressure-dependent phenotypic rescue of GerA channel mutants. Consistently, HP increases IM permeability to water-soluble and membrane-impermeable agents (propidium iodide and formaldehyde), an effect potentiated by GerA, indicating concomitant opening of GerA by HP. Furthermore, modulating IM fluidity via heat activation or decoating altered membrane physical properties and delayed HP-induced germination, establishing the IM as the critical mechanical transducer. Additionally, computational modeling and calculations support faster compression of the IM than of the core under HP, rationalizing the source of tensile stress. Together, our findings establish a novel mechanism of HP-induced GerA activation via the STO model: HP compresses the IM, generates lateral tension, and promotes opening of the GerA ion channel to trigger bacterial spore germination.

Gene transfer agents (GTAs) are phage-derived elements that have evolved repeatedly across diverse prokaryotes, where they drive high-frequency horizontal gene transfer (HGT). Here, we demonstrate that the Rhodobacter capsulatus GTA (RcGTA) tailspike protein, TspA, is a potent biofilm-degrading enzyme. Purified TspA is effective at both preventing initial biofilm formation and clearing established, mature biofilms. Crucially, TspA enhances RcGTA-mediated gene transfer, suggesting that this enzyme facilitates GTA navigation through the extracellular matrix. Unlike the permanently anchored tailspikes of canonical phages, TspA possesses a unique {beta}-sandwich N-terminal domain that enables its dissociation from mature particles and engages in biofilm polysaccharide recognition. Our findings indicate that TspA is an evolutionary adaptation used by GTAs to optimize HGT within complex, densely packed microbial biofilm communities.