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At least 271 records · Page 15

Next-generation large-scale binary protein interaction network for Drosophila melanogaster

Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the ‘FlyBi’ dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.

59 BASIC BIOLOGICAL SCIENCES↗

Diesel Generator Model Parameterization for Microgrid Simulation Using Hybrid Box-Constrained Levenberg-Marquardt Algorithm

Existing generator parameterization methods, typically developed for large turbine generator units, are difficult to apply to small kW-level diesel generators in microgrid applications. Here, this article presents a model parameterization method that estimates a complete set of kW-level diesel generator parameters simultaneously using only load-step-change tests with limited measurement points. This method provides a more cost-efficient and robust approach to achieve high-fidelity modeling of diesel generators for microgrid dynamic simulation. A two-stage hybrid box-constrained Levenberg-Marquardt (H-BCLM) algorithm is developed to search the optimal parameter set given the parameter bounds. A heuristic algorithm, namely Generalized Opposition-based Learning Genetic Algorithm (GOL-GA), is applied to identify proper initial estimates at the first stage, followed by a modified Levenberg-Marquardt algorithm designed to fine tune the solution based on the first-stage result. The proposed method is validated against dynamic simulation of a diesel generator model and field measurements from a 16kW diesel generator unit.

42 ENGINEERING↗

Population genomics and history of speciation reveal fishery management gaps in two related redfish species ( Sebastes mentella and Sebastes fasciatus )

Abstract Understanding the processes shaping population structure and reproductive isolation of marine organisms can improve their management and conservation. Using genomic markers combined with estimation of individual ancestries, assignment tests, spatial ecology, and demographic modeling, we (i) characterized the contemporary population structure, (ii) assessed the influence of space, fishing depth, and sampling years on contemporary distribution, and (iii) reconstructed the speciation history of two cryptic redfish species, Sebastes mentella and S. fasciatus . We genotyped 860 individuals in the Northwest Atlantic Ocean using 24,603 filtered single nucleotide polymorphisms (SNPs). Our results confirmed the clear genetic distinctiveness of the two species and identified three ecotypes within S. mentella and five populations in S. fasciatus . Multivariate analyses highlighted the influence of spatial distribution and depth on the overall genomic variation, while demographic modeling revealed that secondary contact models best explained inter‐ and intragenomic divergence. These species, ecotypes, and populations can be considered as a rare and wide continuum of genomic divergence in the marine environment. This acquired knowledge pertaining to the evolutionary processes driving population divergence and reproductive isolation will help optimizing the assessment of demographic units and possibly to refine fishery management units.

Benestan, Laura M.↗

How to improve scaling from traits to ecosystem processes

Scaling approaches in ecology assume that traits are the main attributes by which organisms influence ecosystem functioning. However, several recent empirical papers have found only weak links between traits and ecosystem functioning, questioning the usefulness of trait-based ecology (TBE). Here, we argue that these studies often suffer from one or more widespread misconceptions. Specifically, these studies often (i) conflict with the conceptual foundations of TBE, (ii) lack theory- or hypothesis-driven selection and use of traits, (iii) tend to ignore intraspecific variation, and (iv) use experimental or study designs that are not well suited to make strong tests of TBE assumptions. Addressing these aspects could significantly improve our ability to scale from traits to ecosystem functioning.

54 ENVIRONMENTAL SCIENCES↗

Thermophilic site-specific recombination system for rapid insertion of heterologous DNA into the Clostridium thermocellum chromosome

Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2–4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 10 3 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6–7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (P clo1313_1194 ) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass.

Biofuels↗

Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

Abstract R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l -arabinose and d -xylose metabolism proceeding through d -arabitol and d -ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d -arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d -arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d -xylose to d -arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d -ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l -ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

59 BASIC BIOLOGICAL SCIENCES↗

A Novel and Secure Pseudovirus Reporter System Based Assay for Neutralizing and Enhancing Antibody Assay Against Marburg Virus

Marburg virus (MARV) is one of the principal members of the filovirus family, which can cause fatal hemorrhagic fever in humans. There are currently no prophylactic and therapeutic drugs on the market, and the high pathogenicity and infectivity of MARV make its research highly dependent on biosafety level 4 conditions, severely hindering the development of vaccines and therapies. Therefore, the development of medicines, such as MARV serological diagnosis, vaccines, and therapeutic antibody drugs, urgently needs a safe, convenient, and biosafety level 2 detection method to measure the neutralizing activity of MARV antibodies. To this end, we report a neutralization assay relying on a Rabies virus (RABV) reverse genetic operating system. We constructed infectious clones carrying the eGFP reporter gene and the full length of the original unmodified MARV GP gene. Based on the critical parameters of phylogenetic analysis, recombinant viruses targeting representative strains in the two major MARV lineages were successfully rescued. These pseudoviruses are safe in mice, and their inability to infect cells after being neutralized by antibodies can be visualized under a fluorescence microscope. We tested the system using the neutralizing antibody MR191. MR191 can significantly block the infection of BSR cells with pseudovirus. We compared it with the traditional lentivirus-type pseudovirus system to verify the system’s credibility and obtained the same results as reported in the literature. In general, we have established a safe and visualized method for evaluating the neutralizing activity of MARV antibodies. Compared with traditional methods, it has the advantages of convenient operation, short cycle, and low cost. It is a candidate method that can replace actual viruses for a neutralization assay.

Bi, Jinhao↗

IMAGINE BioSecurity: Mesocosm-Based Methods to Evaluate Biocontainment Strategies and Impact of Industrial Microbes Upon Native Ecosystems

Project Goals: The Integrative Modeling and Genome-scale Engineering for Biosystems Security (IMAGINE BioSecurity) SFA project seeks to establish an understanding of the behavior of engineered microbes in controlled versus environmental conditions to predictively devise new strategies for responding to biological escape. To this end, the IMAGINE Team has established a plant-soil mesocosm platform to track and quantify the fate of industrial microbes in environmental systems and assess the efficacy of biocontainment constraints upon genetically engineered microbe escape frequency and the impact of industrial microbes upon native ecological microbiomes. Abstract Text: Genetically modified industrial production microbes and their associated bioproducts have emerged as an integral component of a sustainable bioeconomy. However, the rapid development of these innovative technologies raises biosecurity concerns, namely, the risk of environmental escape. Thus, the realization of a bioeconomy hinges not only on the development and deployment of microbial production hosts, but also on the development of secure biosystems and biocontainment designs. Current laboratory-based biocontainment testing systems do not accurately reflect complexities found in natural environments, necessitating an environmentally relevant analysis pipeline that allows for the detection of rare escapees, the effect of associated bio-products, and the impact on native ecologies. To this end, we have developed an approach that utilizes soil mesocosms and integrated systems analyses to evaluate the efficacy of novel biocontainment strategies and to assess the impact of production systems upon terrestrial microbiome dynamics. We demonstrate the utility of this approach by modeling a contamination with industrial microbial chasses versus their biocontained counterparts. Here we demonstrate the broad utility of this system by highlighting findings from both strains of Saccharomyces cerevisiae that are contained with an inducible toxin anti-toxin system, and stains of Escherichia coli that are contained via genomic recoding. The resultant data demonstrate that this system has broad utility across diverse microbial chassis and biocontainment strategies, enables us to track the fate of our contaminating microbe with high sensitivity in the soil, as well as monitor broader impacts of the perturbation on the underlying soil system. The findings presented here support the use of this mesocosm-based approach to assess the environmental impact of industrial microbes and to validate biocontainment strategies.

BASIC BIOLOGICAL SCIENCES,INORGANIC, ORGANIC, PHYS↗

Multi-physics Topology OPtimization and Additive Manufacturing for High-temperature Heat Exchangers

This research significantly advances the understanding of high-temperature heat exchanger design through an integrated approach that combines topology optimization (TO), triply periodic minimal surface (TPMS) structures, additive manufacturing (AM) and thermohydraulic testing. Each of these components contributes uniquely to a unified, high-performance design, fabrication and testing workflow. Topology optimization serves as the foundation of the design methodology by providing a systematic way to determine the most effective material layout for separating hot and cold fluids while maximizing thermal performance. The researchers introduced a novel three-material optimization framework using two density fields to represent hot fluid, cold fluid, and solid domains. This approach enables automated discovery of optimal shapes and flow paths that cannot be intuitively designed, especially under constraints imposed by manufacturing technologies. Furthermore, constraints such as minimal wall thickness and overhang angles were embedded into the optimization process, ensuring that resulting designs are not only thermally efficient but also manufacturable using modern additive techniques. In parallel, the study delves into the use of Gyroid-based TPMS geometries for constructing the core of the heat exchanger. TPMS structures are known for their high surface area, excellent fluid mixing capabilities, and minimal pressure drop characteristics. The researchers applied a data-driven modeling framework using Heteroscedastic Sparse Gaussian Process Regression (HSGPR) combined with genetic algorithms. This allowed for the rapid evaluation and optimization of key geometric parameters such as frequency, iso-value, and phase shift. The result was a set of Gyroid structures tailored for high heat transfer and low flow resistance, demonstrating clear improvements over conventional straight-channel designs. After the designing process, additive manufacturing played a critical role by turning these highly complex, optimized geometries into physical components. Utilizing Laser Powder Bed Fusion (LPBF) with Haynes 282, the study demonstrated the feasibility of fabricating these heat exchangers at high precision. Post-processing methods, including dilation-erosion operations, were applied to ensure local features adhered to self-supporting constraints. The fabricated structures were then subjected to thermohydraulic testing under conditions representative of supercritical CO 2 Brayton cycles, validating the predicted performance and confirming the viability of the full design-to-fabrication pipeline. Finally, thermohydraulic testing across the above studies served as a crucial experimental validation of advanced heat exchanger. Under consistent high-temperature and high-pressure conditions using supercritical CO 2 , the testing demonstrated that both TO and Gyroid-based TPMS designs significantly outperformed conventional straight-channel HXs. The TO design achieved a 115% increase in UA and NTU and a 27.6% boost in gravimetric power density, while the data-driven optimized Gyroid design delivered a 166% increase in UA and NTU and improved effectiveness from 68.7% to 86.1%. These results validate the simulation models, confirm the manufacturability of complex geometries under AM constraints, and provide key insights into design-performance trade-offs, thereby advancing the development of high-efficiency, compact heat exchangers for extreme environments.

36 MATERIALS SCIENCE↗

Classification of bacterial plasmid and chromosome derived sequences using machine learning

Plasmids are important genetic elements that facilitate horizonal gene transfer between bacteria and contribute to the spread of virulence and antimicrobial resistance. Most bacterial genome sequences in the public archives exist in draft form with many contigs, making it difficult to determine if a contig is of chromosomal or plasmid origin. Using a training set of contigs comprising 10,584 chromosomes and 10,654 plasmids from the PATRIC database, we evaluated several machine learning models including random forest, logistic regression, XGBoost, and a neural network for their ability to classify chromosomal and plasmid sequences using nucleotide k-mers as features. Based on the methods tested, a neural network model that used nucleotide 6-mers as features that was trained on randomly selected chromosomal and plasmid subsequences 5kb in length achieved the best performance, outperforming existing out-of-the-box methods, with an average accuracy of 89.38% ± 2.16% over a 10-fold cross validation. The model accuracy can be improved to 92.08% by using a voting strategy when classifying holdout sequences. In both plasmids and chromosomes, subsequences encoding functions involved in horizontal gene transfer—including hypothetical proteins, transporters, phage, mobile elements, and CRISPR elements—were most likely to be misclassified by the model. This study provides a straightforward approach for identifying plasmid-encoding sequences in short read assemblies without the need for sequence alignment-based tools.

59 BASIC BIOLOGICAL SCIENCES↗

Intra‐species genetic variability drives carbon metabolism and symbiotic host interactions in the ectomycorrhizal fungus Pisolithus microcarpus

Summary Ectomycorrhizal (ECM) fungi are integral to boreal and temperate forest ecosystem functioning and nutrient cycling. ECM fungi, however, originate from diverse saprotrophic lineages and the impacts of genetic variation across species, and especially within a given ECM species, on function and interactions with the environment is not well understood. Here, we explore the extent of intra‐species variation between four isolates of the ECM fungus Pisolithus microcarpus , in terms of gene regulation, carbon metabolism and growth, and interactions with a host, Eucalyptus grandis . We demonstrate that, while a core response to the host is maintained by all of the isolates tested, they have distinct patterns of gene expression and carbon metabolism, resulting in the differential expression of isolate‐specific response pathways in the host plant. Together, these results highlight the importance of using a wider range of individuals within a species to understand the broader ecological roles of ECM fungi and their host interactions.

Plett, Krista L.↗

Higher Wood Density Lowers Feedstock Cost and Has Minimal Impact on Biomass Conversion to Biofuels

Poplar and other woody feedstocks have the potential to provide up to 200 million tons of biomass per year that can be converted to liquid fuels. Most forestry strategies that aim to increase biomass productivity per hectare rely on short rotation plantations of fast-growing varieties. The improvement of the wood density as a key trait itself has largely been overlooked. We evaluated natural variation in wood density across a population of genetically diverse Populus trichocarpa trees grown in a common garden. Wood density varies greatly within this population but is heritable; higher wood density was not systematically associated with reduced growth, challenging assumptions of a trade-off between wood density and biomass accumulation. Furthermore, denser wood led to significant improvements throughout the supply chain including lowering biomass production and transportation costs. Higher density did not correlate with changes in biomass composition. Density did not impact bioconversion in the two feedstock-to-fuel pipelines tested (pretreatment by ionic liquids and fermentation to bisabolene or soaking in aqueous ammonia and fermentation to ethanol) on a representative subset of poplars. These findings highlight wood density as a promising breeding target for accelerating the development of high-yielding, conversion-efficient bioenergy crops and as an avenue for increasing landuse efficiency and reducing biomass transportation cost.

09 BIOMASS FUELS↗

Coding and noncoding variants in EBF3 are involved in HADDS and simplex autism

Abstract Background Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737. Results We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10 −3 ), and combined dataset (p = 1.1 × 10 −4 ). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10 −35 , loss-of-function p = 2.26 × 10 −13 ) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10 −6 , OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia. Conclusions In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.

59 BASIC BIOLOGICAL SCIENCES↗

Fecal microbiota transplantation ameliorates type 2 diabetes via metabolic remodeling of the gut microbiota in db/db mice

Gut microbiome (GM) deregulation has been implicated in major conditions such as obesity and type 2 diabetes (T2DM). Our previous prospective study indicated that fecal microbiota transplantation (FMT) successfully improved patients with T2DM. We hypothesized that FMT may be a potential therapeutic method for T2DM, but its precise mechanisms in T2DM remains to be elucidated. Eight db/m mice were FMT donors and control mice, and 16 genetically diabetic db/db mice were equally divided into two groups (db/db+phosphate-buffered saline (PBS) group, db/db+FMT group). The db/db+FMT group was administered fresh fecal suspension (0.2 mL/mice) daily for 4 weeks. Analysis of the GM and serum metabolome was carried out by 16S ribosomal RNA sequencing and liquid chromatogram-mass spectrometry, respectively. Effects of FMT on the gut barrier and pancreas were assessed using protein assays, messenger RNA, immunohistology and clinical indicators testing. Our results showed that FMT treatment of db/db mice relieves a series of clinical indicators, including fasting plasma glucose, serum insulin and oral glucose tolerance test among others. Compared with non-diabetic control mice, db/db+PBS mice exhibited decreased abundance of Ruminococaceae, Porphyromonadaceae and increased abundance of Rikenellaceae and Lactobacillaceae. FMT treatment reversed this effect on the microbiome. Eleven metabolites were changed between the db/db+PBS and db/db+FMT groups. Correlation analysis showed that the structural changes of the GM were correlated with host metabolite levels. We further showed that FMT treatment of db/db mice improved intestinal barrier function, reduced inflammation and caused an alteration in the number of circulating immune cells. FMT-mediated changes in the GM, serum metabolites, intestinal epithelial barrier, inflammation and circulating immune cells play an important role in the efficacy of FMT on T2DM disease progression.

60 APPLIED LIFE SCIENCES↗

Optimization of a High Bunch Charge ERL Injection Merger for PERLE

Delivery of high charge electron bunches into the main loop of an ERL (energy recovery linac) while preserving the emittance is challenging. This is because at the typical injection momentum, space charge forces still have a significant effect on the beam dynamics. In this work we consider the design of the merger for PERLE, an ERL test facility to be based at IJCLab in France. Previous simulations have shown that the baseline DC gun based injector can achieve the required emittance at the booster linac exit. The quality of the 500 pC bunches must then be preserved with space charge through the merger at total beam energy of 7 MeV keeping the emittance below 6 mm mrad. The beam dynamics in the merger were simulated using the code OPAL and optimised using a genetic algorithm. Three possible merger schemes were investigated. The goal of the optimisation was to minimise the emittance growth while also achieving the required Twiss parameters to match onto the spreader at the main linac exit. A three dipole solution is then examined in more detail.

Hounsell, B.↗

Dinitrogen Binding and Functionalization from a Low-Coordinate Alkynyliron Complex

Alkynyl complexes of low-coordinate transition metals offer a sterically open environment and interesting bonding opportunities. Here, in this work, we explore the capacity of iron(I) alkynyl complexes to bind N 2 and isolate a N 2 complex including its X-ray crystal structure. Silylation of the N 2 complex gives an isolable, formally iron(IV) complex with a disilylhydrazido(2−) ligand, but natural bond orbital analysis indicates that an iron(II) formulation is preferable. The structure of this compound is similar to an earlier reported phenyl complex in which phenyl migration forms a new N–C bond, but the alkynyl group does not migrate. DFT calculations are used to test the possible reasons why the alkynyl is resistant to migration, and these show that the large Fe–C bond energy in the alkynyl complex is a factor that could contribute to the lack of migration.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Cell culture systems have greatly expanded our understanding of how bacterial pathogens target signaling pathways to manipulate the host and cause infection. Advances in genetic engineering have allowed for the creation of fluorescent protein readouts within signaling pathways, but these techniques have been underutilized in pathogen biology. Here, we genetically engineered a lung cell line with fluorescent reporters for extracellular signal-related kinase (ERK) and the downstream transcription factor FOS-related antigen 1 (Fra1) and evaluated signaling after inoculation with pathogenic and non-pathogenic bacteria. Cells were inoculated with 100 colony-forming units of Acinetobacter baylyi, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus agalactiae, or Staphylococcus epidermidis and imaged in a multi-mode reader. The alamarBlue cell viability assay was used as a reference test and showed that pathogenic P. aeruginosa induced significant (p < 0.05) cell death after 8 h in both wild-type and engineered cell lines compared to non-pathogenic S. epidermidis. In engineered cells, we found that Fra1 signaling was disrupted in as little as 4 h after inoculation with bacterial pathogens compared to delayed disruption in signaling by non-pathogenic S. epidermidis. Overall, we demonstrate that low levels of pathogenic versus non-pathogenic bacteria can be rapidly and sensitively screened based on ERK-Fra1 signaling

59 BASIC BIOLOGICAL SCIENCES↗

Machine learning-enabled phenotyping for GWAS and TWAS of WUE traits in 869 field-grown sorghum accessions

Abstract Sorghum (Sorghum bicolor) is a model C4 crop made experimentally tractable by extensive genomic and genetic resources. Biomass sorghum is studied as a feedstock for biofuel and forage. Mechanistic modeling suggests that reducing stomatal conductance (gs) could improve sorghum intrinsic water use efficiency (iWUE) and biomass production. Phenotyping to discover genotype-to-phenotype associations remains a bottleneck in understanding the mechanistic basis for natural variation in gs and iWUE. This study addressed multiple methodological limitations. Optical tomography and a machine learning tool were combined to measure stomatal density (SD). This was combined with rapid measurements of leaf photosynthetic gas exchange and specific leaf area (SLA). These traits were the subject of genome-wide association study and transcriptome-wide association study across 869 field-grown biomass sorghum accessions. The ratio of intracellular to ambient CO2 was genetically correlated with SD, SLA, gs, and biomass production. Plasticity in SD and SLA was interrelated with each other and with productivity across wet and dry growing seasons. Moderate-to-high heritability of traits studied across the large mapping population validated associations between DNA sequence variation or RNA transcript abundance and trait variation. A total of 394 unique genes underpinning variation in WUE-related traits are described with higher confidence because they were identified in multiple independent tests. This list was enriched in genes whose Arabidopsis (Arabidopsis thaliana) putative orthologs have functions related to stomatal or leaf development and leaf gas exchange, as well as genes with nonsynonymous/missense variants. These advances in methodology and knowledge will facilitate improving C4 crop WUE.

54 ENVIRONMENTAL SCIENCES↗