Algal Research

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI

Fluorescent reporters cover a wide range of applications in both basic and applied research. Whether a study involves microscopic imaging to study (co)-localization of proteins, FRET, biosensing, or quantifying gene expression, fluorophores are attractive reporter candidates due to their relatively straightforward in vivo readout. For microbiological applications, a wide variety of fluorescent proteins with varying excitation and emission wavelengths, brightness levels, and maturation times are available. Careful consideration is required when selecting from this large suite of proteins, especially when choosing multiple fluorophores. This is further complicated in phototrophic organisms, which exhibit strong autofluorescence, especially towards the red part of the spectrum, effectively eliminating common candidates such as mCherry. In this study, the specific properties and performance of a selection of fluorescent proteins are systematically evaluated against the background of photosynthetic pigment-derived autofluorescence in the cyanobacterium Synechocystis sp. PCC 6803. Specific readouts of different combinations of fluorescent proteins are also analyzed using high-throughput methods, namely plate reader fluorescent scans and single-cell flow cytometry to quantify fluorescence. The ultimate goal is to assess each fluorescent protein with regard to: 1.) Its ability to be discerned from cyanobacterial autofluorescence. 2.) Its compatibility with other fluorophores in this context. 3.) Its overall suitability in cyanobacterial research. Several highly suitable fluorescent proteins for use in cyanobacteria are identified, including mTagBFP2, mNeonGreen and mScarlet-I and suitable combinations, covering nearly the whole spectrum of visible light. This study expands the knowledge and toolset for current and future researchers and uncovers a whole spectrum of possibilities for fluorescent protein selection in cyanobacterial cell biology.

Fast cytoplasmic streaming enables extensive chloroplast movements in the giant cells of unicellular, siphonous macroalgae. Here, we studied chloroplast movements in two such algae: the Dasycladalean Acetabularia acetabulum and the Bryopsidales Bryopsis sp.. We hypothesised that chloroplast movements function as a protective avoidance mechanism under excess light, particularly in Bryopsis sp., which lacks capacity for fast induction of photoprotective non-photochemical quenching (NPQ) and state transitions. In addition, we also investigated whether chloroplast movements are involved in responses to wounding and herbivory. The movements were studied by light microscopy, photography and pulse modulated chlorophyll a fluorescence quenching analysis. Chemical inhibitors of actin polymerization and microtubules assembly were used to confirm that the observed effects were active responses controlled by the cytoskeleton. A. acetabulum responded to high light by reversible chloroplast aggregation, probed by macro-imaging; and chemical inhibition of chloroplast movements led to an enhancement of Photosystem II photoinhibition, as probed by the fluorescence parameter FV/FM. No chloroplast movements were observed in Bryopsis sp. in response to high light. In A. acetabulum, wounding caused either by cutting or due to feeding by the sap-sucking sea slug Elysia timida triggered aggregation of chloroplasts within minutes of incurring the damage. Interestingly, the aggregation also occurred in intact cells away from the cutting site. Furthermore, the addition of media collected from the vicinity of cut algae was sufficient to induce chloroplast aggregation in intact algae, suggesting that water-borne cues or signals triggered the aggregation response in A. acetabulum. Bryopsis sp., however, responded to cutting by only local chloroplast aggregation. The relevance of chloroplast movements in protection against both abiotic and biotic stressors in A. acetabulum, and the potential reasons behind the different defence strategies of the algae, are discussed.

Chlamydomonas reinhardtii is a model green alga extensively used to study photosynthesis and cilia using molecular biology and genetics. Electroporation is a very common technique to transform DNA into the nuclear genome, which is essential to generate mutant collections and express transgenes. Here, we describe a simple, fast, and efficient protocol to transform strains with an intact cell wall. It achieves a good transformation efficiency without cell wall digestion or use of commercial kits and is compatible with the widely available Gene Pulser electroporation system. Key featuresO_LIHigh transformation efficiency of Chlamydomonas reinhardtii strains with an intact cell wall. C_LIO_LIFaster than currently available electroporation protocols. C_LI

Fermentation of C1 gases is an emerging technology where waste gases are bio converted into value-added products. This study navigates the gas fermentation potential of Gordonia rubripertincta to produce carotenoids. The crucial carbon monoxide dehydrogenase (CODH) enzyme, necessary for gas uptake by the microbe, was found to be present in G. rubripertincta through blastp on NCBI website. The organism was then used for gas fermentation experiments in a continuous stirred tank reactor (CSTR) in different modes of reactor operation resulting in the production of about 500 mg pigment/g WCW (wet cell weight). Two important reactor parameters, molybdenum content and pH, were optimized for enhanced carotenoid production. Overall, G. rubripertincta was observed to be an efficient candidate organism for C1 gas fermentation. KEY HIGHLIGHTSO_LIGordonia rubripertincta synthesises aerobic carbon monoxide dehydrogenase enzyme. C_LIO_LIIt is a potential gas fermenting microbe that gives carotenoids as product. C_LIO_LIThe gas uptake efficiency of the microbe is more in fed-batch discontinued mode. C_LIO_LIIn FB-D, the resultant carotenoids are 500+9 mg/g wet cell weight (WCW). C_LIO_LIMo/pH of 20 mg/7.0 resulted in highest carotenoids, i.e., 134+41 mg/g WCW. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/722808v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@8b1185org.highwire.dtl.DTLVardef@2b6f90org.highwire.dtl.DTLVardef@1a9697dorg.highwire.dtl.DTLVardef@14c9dc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Edible seaweed has the potential to become a valuable marine resource for food applications due to its potential health benefits and ecological sustainability. The brown seaweed Alaria esculenta is rich in essential minerals, vitamins, and dietary fibers, making it a nutritious food source. Fermentation, as a traditional preservation method, can enhance seaweed shelf-life and be useful for the development of new foods/ beverages. In this study, the effects of fermentation of A. esculenta, by the lactic acid bacterium (LAB) Lactiplantibacillus plantarum, on the nutritional profile, and the content of potentially toxic elements, was investigated. L. plantarum was successfully cultivated on A. esculenta using two modes of operation, submerged (SmF) and solid-state fermentation (SSF), resulting in production of cells and lactic acid, and reduction of the pH to below 4.3 within 3 days, which was not achieved in parallel spontaneous fermentations using indigenous seaweed microbiota. A. esculenta s macro-nutritional profile was altered, reducing mannitol but increasing fucose and glucose content (after acid hydrolysis) while also concentrating the protein content. LAB fermentation significantly increased the concentration of antioxidant phenolic compounds, such as phloroglucinol, syringic acid, and epicatechin, compared to untreated samples. However, lipophilic compounds like carotenoids decreased after both spontaneous and LAB-fermentation. A reduction in total mineral content was observed after LAB fermentation and water soaking, and SmF with L. plantarum effectively reduced arsenic and iodine levels. Overall, fermentation using L. plantarum showed potential as a bio-preservation method for the edible brown seaweed, A. esculenta, improving its nutritional profile and enhancing food safety.

As one of the earliest-diverging multicellular eukaryotic lineages, the bladed Bangiales (Rhodophyta) possess a deep evolutionary history with a central role in the multi-billion-dollar global seaweed aquaculture industry. Although North Atlantic representatives are emerging candidates for regional mariculture, the scarcity of high-quality genomic resources for these taxa hinders both fundamental research and commercial optimization. To address this, we present the first chromosome-level genome assemblies for two native European species: Porphyra dioica (150.44 Mbp) and Porphyra linearis (95.22 Mbp). By integrating Oxford Nanopore Technologies (ONT) long-read sequencing with Hi-C proximity ligation, we generated highly contiguous nuclear genomes resolved into five chromosomes. Structural gene models were predicted through the BRAKER3 pipeline, identifying 12,548 and 10,382 protein-coding genes for P. dioica and P. linearis, respectively. Subsequent homology-based functional annotation characterized 57.4% and 59.8% of these predicted proteins. Supplemented by circularized organellar genomes, these reference genomes provide a critical framework for future research, enabling comparative studies of Atlantic-Pacific divergence and facilitating the development of selective breeding programs for sustainable European aquaculture.

Freshwater algae represent an underexplored source of naturally occurring bioactive metabolites with potential applications in pharmaceutical and biomedical research. This study investigated the phytochemical composition, antioxidant capacity, and preliminary cytotoxic potential of ethanolic and n-hexane extracts of freshwater algal species collected at Jilani Park, Lahore, Pakistan. Algal species were identified morphologically by Dr. Ghazal Yasmeen (Institute of Botany, Punjab University, Lahore). Extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and qualitative phytochemical screening. Antioxidant activity was evaluated using DPPH radical scavenging, hydrogen peroxide scavenging, and reducing power assays. Cytotoxic potential was assessed using MTT and cell adhesion assays on HeLa and SF767 cell lines as preliminary indicators of bioactivity. GC-MS analysis identified 25 compounds, including sterols, fatty acid esters, terpenoids, phenolic compounds, and volatile metabolites. Phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, and terpenoids in the extracts. Among the tested extracts, the n-hexane fraction demonstrated comparatively higher antioxidant activity across multiple assays. Ethanolic extracts showed moderate reductions in HeLa cell viability, whereas limited effects were observed in SF767 cells. These findings suggest that freshwater algae are promising natural reservoirs of antioxidant metabolites with potential relevance for future isolation and characterization of bioactive compounds for biomedical applications. Further purification and mechanistic studies are required to identify specific active constituents.

BackgroundThe use of autologous or allogeneic cell therapies has now entered to the clinical practice in several fields of medicine, especially in oncology and hematology. From this regard, 2D-cell manufacturing is complex and costly and bioreactors have attracted major interest for efficient and cost-effective mass production of cells. Bioreactors have several advantages such as homogeneous repartition of nutrients and gas, control of all culture parameters and increased yield. However, the important shear stress generated by those bioreactors is an important disadvantage as it can affect cell survival or cell quality. This important shear stress is the result of the mixing method using either blades (used in stirred-tanked bioreactors) or gas bubbles (used in airlift bioreactors). Another downside of the use of bioreactors is the difficulty to scale-up. As the volume increases, the shear stress generated by blades radically increases leading to cell death and a decrease of cell quality. DescriptionIn this study, we describe a bioreactor developed using a different mixing method effectively reducing the shear stress and facilitating scale-up. This bladeless method uses an inclination of the bioreactor as well as rotation to mix fluids in a container. Here we described different steps that led to the adaptation of this bioreactor, initially developed for fragile microalgae culture, for mammalian cell culture amplification. The bioreactor was tested to amplify a natural killer (NK) cell line NK92 which is an IL-2 dependent cell line used in clinical trials for cancer therapy. We have tested the influence of 1-The number of cells seeded; 2-The influence of the rotation speed on cell growth and viability; 3-The influence of the bioreactor angle on the above parameters; 4-The duration of the culture. ResultsCells were initially seeded at 2.5.105 / ml in a volume of 380 ml. According to the rotation speed of 15, 30, 45 and 60 rpm, we have observed an increase of cell numbers at day 3 (3-fold), day 5 (7-fold) and day 7 (10-fold) compared to seeding, the best expansion being obtained at day 7 with a rotation speed of 45 rpm. The optimal angle of rotation was found to be 3 degree, with an optimal amplification at day 7 versus day 3 (p < 0.01). The viability was also found to be optimal in the latter condition. ConclusionsThese preliminary results demonstrate that NK92 cells could be amplified using this bioreactor. In the best tested condition, neither cell viability nor cell growth was impacted. These results strongly suggest the potential use of this device in future clinically applicable conditions.

1.Abiotic stresses like nitrogen deficiency and soil salinity are major factors contributing to low crop yields. The use of selective biofertilizers alleviates both types of stress. In this study, we investigated the biofertilizer activity and plant growth-promoting properties (PGP) of Rhodococcus jialingiae RS1 through cytosolic proteome remodelling. We cultured RS1 under two conditions, i) without and ii) with 6% NaCl, in nitrogen-deficient defined Burks medium. Under dual stress of nitrogen limitation and salt stress, Orbitrap LC-MS/MS proteomics revealed one-quarter of the proteome remodelling, particularly the upregulation of ribosomal synthesis and protein repair systems. As expected, we found high expression of EctC, an ectoine synthase, a key enzyme in osmolyte biosynthesis. Additionally, ribosomal and translational-associated factors, including RpsL, RpsS, RpsT, RpsR1, RplV, RplL, RplA, and elongation factor Tuf, were highly expressed, suggesting enhanced translational fidelity under dual stress. High levels of DNA protection protein, Dps suggest dual stress may lead to DNA damage. Upregulation of chaperones, environmental sensors (KinE), and redox transcriptional factors like WhiB3, Hsp18, AhpC, and MetE suggests protein misfolding and oxidative stress. Metabolic modulations were evident through high expression of IlvA, NAD-dependent glutamate dehydrogenase, lipid/envelope-remodelling enzymes, cutinase/esterases, lipases, endopeptidases like NlpC/P60 and transport systems. In contrast, proteins involved in urease structural components (urea-G), nitrogen regulators and ammonium transporters (GlnK and Amt) were downregulated. Dual stress may lead to an energy crisis, prompting strategic shifts away from high-ATP-dependent ureolytic nitrogen-scavenging pathways towards lower-energy nitrogen-assimilating routes, such as IlvA-mediated deamination and NAD-dependent glutamate dehydrogenation. Genetic manipulations of the above-mentioned genes or their homologues across the genera of microbes, plants, and crops may enhance resilience to abiotic stresses. Our studies reveal stress-responsive genes and biochemical pathways that could be used to improve transgenic efficacy in nitrogen-limited, saline soil and other (a)biotic stresses. Global Proteome Profiling of Rhodococcus jialingiae RS1 to Develop Transgenics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/724437v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1719d80org.highwire.dtl.DTLVardef@1b6b59org.highwire.dtl.DTLVardef@24d367org.highwire.dtl.DTLVardef@1b33224_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.

The fast-growing cyanobacterium Synechococcus sp. PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.

Manufacturing and storage processes can expose microbes to oxidative stress, reducing viability and limiting their use in biotechnological applications. Here, we evaluate graphene quantum dots (GQDs) containing hydroxyl and carboxyl groups as protective additives that mitigate peroxide-induced oxidative stress in Escherichia coli. GQDs did not adversely affect bacterial growth under basal conditions and restored growth in the presence of hydrogen peroxide. Using the membrane-partitioning fluorescent probe C11-BODIPY, we found that GQDs reduced peroxide-induced oxidation in bacterial membranes. We further used redox-sensitive roGFP2 probes to monitor intracellular oxidative stress and found that GQDs suppressed intracellular hydrogen peroxide accumulation and attenuated disruption of glutathione redox homeostasis. Together, these results show that GQDs protect bacteria by limiting peroxide-driven oxidative damage at both membrane and intracellular levels. This work supports the potential use of GQDs as protective additives for microbial formulations that are susceptible to oxidative stress.

Seaweed cultivation has recently gained increased attention in North-West Europe as a sustainable source of biomass for biobased products. However, yields need to increase to make the seaweed sector economically viable. To achieve this, higher yielding varieties can be bred but this requires variation for yield and yield-related traits among genotypes. To reliably select high-yielding genotypes, an understanding is required of how both within-farm and between-farm environmental differences affect phenotypes and how to identify simple and reliable proxies for yield. In this study we evaluated growth of nine Saccharina latissima genotypes on two farms, 12 km apart, within the same season. We observed a threefold difference in yield among genotypes, demonstrating the potential for improvement through selection and breeding. Blade thickness and blade size-related traits were strongly correlated with yield, highlighting their potential to serve as rapid and non-destructive proxies for yield, thereby accelerating selection. Furthermore, we demonstrated the importance of adequate replication in farm trials to improve genotype performance estimation by correcting for within-farm spatial variation. Moreover, phenotypic variation was most explained by the genotype and environment, highlighting the importance of both genotype and site selection. Although genotype by environment interactions (GxE) were significant, its contributions were small, indicating stable genotype ranking across farms. Overall, these results are promising for breeding improved S. latissima as it indicates that genotype performance is consistent across close by locations and that local S. latissima populations harbour substantial phenotypic variation that can be used to breed for increased yield. Highlights- Local genetic resources harbour substantial variation in yield and morphology for breeding. - Minor GxE allows for breeding across farms. - Blade thickness and blade size related traits are good predictors of yield. - Correction for on-farm spatial variation improves genotype performance estimation.

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

Coordination among cell growth, chloroplast expansion, and organelle genome dynamics is fundamental to algal physiology, yet its regulation remains unclear. We used time-resolved single-cell analyses to examine scaling relationships among cell size, chloroplast volume, nuclear dynamics, and nucleoid organization in Desmodesmus communis and Chlamydomonas reinhardtii under normal conditions and after inhibition of chloroplast DNA replication with nalidixic acid (NAL). Under control conditions, both species showed coordinated scaling among cell, chloroplast, and nuclear size, while nucleoid dynamics were driven mainly by changes in number. NAL disrupted these relationships in a species- and time-dependent manner. In C. reinhardtii, prolonged treatment uncoupled chloroplast and nuclear growth from cell expansion and led to fewer, enlarged nucleoids, consistent with impaired replication. In contrast, D. communis largely maintained coordinated scaling, with effects mainly limited to reduced nucleoid proliferation and delayed division. Temporal analyses indicated that NAL primarily affected nucleoid replication and segregation, with secondary consequences for chloroplast growth and cell-cycle progression. These findings identify chloroplast genome dynamics as a regulatory link between organelle growth and cell division.

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days under mildly acidic conditions to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (10-20 g/L), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (30-40 g/L; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis indicated that under high-lactate conditions up to 30% of lactate was redirected toward propionate and acetate formation, likely via the acrylate pathway. Microbial community analysis revealed a stable dominance of Caproiciproducens spp., that could be correlated to valerate production. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems. HighlightsValerate was the dominant product, reaching up to 110 mM. Lactate:propionate ratios drive product selectivities. High lactate concentrations activated in situ propionate formation pathways. Caproiciproducens dominance was associated with sustained valerate production.

Phenotypic plasticity can buffer the potential fitness consequences of environmental change, yet limited understanding of its genetic basis constrains its application to predicting population response to future climates. Hybrid zones provide powerful systems to study the genetic basis of plasticity because admixture can create novel allele combinations that generate new reaction norms for selection to act upon. Here, we combine two clonally replicated common gardens of Populus trichocarpa x P. balsamifera genotypes with whole-genome resequencing to identify genetic variation underlying plasticity for physiological traits. Admixed genotypes exhibited broader, and in some cases novel, reaction norms relative to parental genotypes, particularly for key stomatal traits. Admixture mapping of genotype-specific reaction norms identified ten candidate genes on chromosome 15, including TWIST, associated with plasticity in adaxial stomatal occurrence and density. Using random forest models, we projected allele-specific responses to climate warming within the hybrid zone to link genetic variation in plasticity with predicted warming. Random forest models forecast that future climates would favor P. trichocarpa alleles at TWIST, while P. balsamifera alleles would be maintained in heterozygous genotypes. These results suggest that hybridization can expand reaction norms and maintain genetic variation that may facilitate rapid phenotypic response needed to adapt to climate change.

Many hermaphroditic species increase outcrossing rates by partitioning reproduction so that male and female organs mature at different times, a phenomenon known as dichogamy. Previous work has documented that dichogamy in pecan trees is governed by the Mendelian super-gene "G-locus"; however, its non-recombinant sex chromosome-like architecture has impeded quantitative genetic exploration and candidate gene discovery. Here, we probe the genetic drivers of the G-locus through a pangenome-integrated quantitative genomics experiment. We first provide one of the clearest examples to date of mapping bias, where a linear reference-based GWAS discovered 66 off-target peaks while mapping with a pangenome graph reference resolved the known single Mendelian locus. Across six new genome assemblies, the fully haplotype phased G-locus QTL spanned 223-491kb and included 25 candidate gene families. The strongest candidate gene encoded a MATE efflux protein and had dominant allele-specific action during male flower developmental stages. Combined, these candidates and genomic resources provide a powerful foundation for breeding and optimal dichogamy phenotype engineering for future pecan orchards.