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At least 397 records · Page 22
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Computer vision models enable mixed linear modeling to predict arbuscular mycorrhizal fungal colonization using fungal morphology

Abstract The presence of Arbuscular Mycorrhizal Fungi (AMF) in vascular land plant roots is one of the most ancient of symbioses supporting nitrogen and phosphorus exchange for photosynthetically derived carbon. Here we provide a multi-scale modeling approach to predict AMF colonization of a worldwide crop from a Recombinant Inbred Line (RIL) population derived from Sorghum bicolor and S. propinquum . The high-throughput phenotyping methods of fungal structures here rely on a Mask Region-based Convolutional Neural Network (Mask R-CNN) in computer vision for pixel-wise fungal structure segmentations and mixed linear models to explore the relations of AMF colonization, root niche, and fungal structure allocation. Models proposed capture over 95% of the variation in AMF colonization as a function of root niche and relative abundance of fungal structures in each plant. Arbuscule allocation is a significant predictor of AMF colonization among sibling plants. Arbuscules and extraradical hyphae implicated in nutrient exchange predict highest AMF colonization in the top root section. Our work demonstrates that deep learning can be used by the community for the high-throughput phenotyping of AMF in plant roots. Mixed linear modeling provides a framework for testing hypotheses about AMF colonization phenotypes as a function of root niche and fungal structure allocations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

Abstract Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Sustainable strategies to achieve industrial ethanol titers from different bioenergy feedstocks: scale-up approach for better ethanol yield

Hydrothermal pretreatment is a promising approach to lignocellulosic biomass processing for enzymatic hydrolysis and high-yield bioethanol fermentation, as it reduces downstream inhibitor content and the amount of toxic byproducts generated. In this paper, the ethanol yield and productivity of an engineered xylose-fermenting strain of Saccharomyces cerevisiae were tested on lignocellulosic hydrolysates produced with varying citrate buffer concentration, solid loading, supplemental nitrogen source, and feedstock of origin, and a semi-integrated bioprocess which integrates enzymatic hydrolysis and bioethanol fermentation was developed. The greatest ethanol yields (g p /g s ) of 0.490 ± 0.008, 0.460 ± 0.001, 0.420 ± 0.002 and 0.410 ± 0.002 were obtained from bioenergy sorghum (BES), Miscanthus × giganteus (MG), energy cane (EC), and oilcane (OC), respectively. In addition, an equivalent of 291 L, 253.54 L, 257.8 L, and 260.3 L of bioethanol were produced per ton of BES, MG, EC, and OC, respectively, by using urea as a nitrogen source in a bioreactor.

09 BIOMASS FUELS↗
DOE OSTI2023

Similar photosynthetic but different yield responses of C 3 and C 4 crops to elevated O 3

The deleterious effects of ozone (O 3 ) pollution on crop physiology, yield, and productivity are widely acknowledged. It has also been assumed that C 4 crops with a carbon concentrating mechanism and greater water use efficiency are less sensitive to O 3 pollution than C 3 crops. This assumption has not been widely tested. Therefore, we compiled 46 journal articles and unpublished datasets that reported leaf photosynthetic and biochemical traits, plant biomass, and yield in five C 3 crops (chickpea, rice, snap bean, soybean, and wheat) and four C 4 crops (sorghum, maize, Miscanthus × giganteus , and switchgrass) grown under ambient and elevated O 3 concentration ([O 3 ]) in the field at free-air O 3 concentration enrichment (O 3 -FACE) facilities over the past 20 y. When normalized by O 3 exposure, C 3 and C 4 crops showed a similar response of leaf photosynthesis, but the reduction in chlorophyll content, fluorescence, and yield was greater in C 3 crops compared with C 4 crops. Additionally, inbred and hybrid lines of rice and maize showed different sensitivities to O 3 exposure. This study quantitatively demonstrates that C 4 crops respond less to elevated [O 3 ] than C 3 crops. This understanding could help maintain cropland productivity in an increasingly polluted atmosphere.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Three founding ancestral genomes involved in the origin of sugarcane

Modern sugarcane cultivars (Saccharum spp.) are high polyploids, aneuploids (2n = ~12 = ~120) derived from interspecific hybridizations between the domesticated sweet species Saccharum officinarum and the wild species S. spontaneum. To analyse the architecture and origin of such a complex genome, we analysed the sequences of all 12 hom(oe)ologous haplotypes (BAC clones) from two distinct genomic regions of a typical modern cultivar, as well as the corresponding sequence in Miscanthus sinense and Sorghum bicolor, and monitored their distribution among representatives of the Saccharum genus. The diversity observed among haplotypes suggested the existence of three founding genomes (A, B, C) in modern cultivars, which diverged between 0.8 and 1.3 Mya. Two genomes (A, B) were contributed by S. officinarum; these were also found in its wild presumed ancestor S. robustum, and one genome (C) was contributed by S. spontaneum. These results suggest that S. officinarum and S. robustum are derived from interspecific hybridization between two unknown ancestors (A and B genomes). The A genome contributed most haplotypes (nine or ten) while the B and C genomes contributed one or two haplotypes in the regions analysed of this typical modern cultivar. Interspecific hybridizations likely involved accessions or gametes with distinct ploidy levels and/or were followed by a series of backcrosses with the A genome. The three founding genomes were found in all S. barberi, S. sinense and modern cultivars analysed. None of the analysed accessions contained only the A genome or the B genome, suggesting that representatives of these founding genomes remain to be discovered. This evolutionary model, which combines interspecificity and high polyploidy, can explain the variable chromosome pairing affinity observed in Saccharum. It represents a major revision of the understanding of Saccharum diversity.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

MINE: a new way to design genetics experiments for discovery

Abstract The Maximally Informative Next Experiment or MINE is a new experimental design approach for experiments, such as those in omics, in which the number of effects or parameters p greatly exceeds the number of samples n (p > n). Classical experimental design presumes n > p for inference about parameters and its application to p > n can lead to over-fitting. To overcome p > n, MINE is an ensemble method, which makes predictions about future experiments from an existing ensemble of models consistent with available data in order to select the most informative next experiment. Its advantages are in exploration of the data for new relationships with n < p and being able to integrate smaller and more tractable experiments to replace adaptively one large classic experiment as discoveries are made. Thus, using MINE is model-guided and adaptive over time in a large omics study. Here, MINE is illustrated in two distinct multiyear experiments, one involving genetic networks in Neurospora crassa and a second one involving a genome-wide association study in Sorghum bicolor as a comparison to classic experimental design in an agricultural setting.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Poisson hurdle model-based method for clustering microbiome features

Abstract Motivation High-throughput sequencing technologies have greatly facilitated microbiome research and have generated a large volume of microbiome data with the potential to answer key questions regarding microbiome assembly, structure and function. Cluster analysis aims to group features that behave similarly across treatments, and such grouping helps to highlight the functional relationships among features and may provide biological insights into microbiome networks. However, clustering microbiome data are challenging due to the sparsity and high dimensionality. Results We propose a model-based clustering method based on Poisson hurdle models for sparse microbiome count data. We describe an expectation–maximization algorithm and a modified version using simulated annealing to conduct the cluster analysis. Moreover, we provide algorithms for initialization and choosing the number of clusters. Simulation results demonstrate that our proposed methods provide better clustering results than alternative methods under a variety of settings. We also apply the proposed method to a sorghum rhizosphere microbiome dataset that results in interesting biological findings. Availability and implementation R package is freely available for download at https://cran.r-project.org/package=PHclust. Supplementary information Supplementary data are available at Bioinformatics online.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

DCT4 —A New Member of the Dicarboxylate Transporter Family in C 4 Grasses

Malate transport shuttles atmospheric carbon into the Calvin–Benson cycle during NADP-ME C 4 photosynthesis. Previous characterizations of several plant dicarboxylate transporters (DCT) showed that they efficiently exchange malate across membranes. Here, we identify and characterize a previously unknown member of the DCT family, DCT4, in Sorghum bicolor. We show that SbDCT4 exchanges malate across membranes and its expression pattern is consistent with a role in malate transport during C 4 photosynthesis. SbDCT4 is not syntenic to the characterized photosynthetic gene ZmDCT2, and an ortholog is not detectable in the maize reference genome. We found that the expression patterns of DCT family genes in the leaves of Zea mays, and S. bicolor varied by cell type. Our results suggest that subfunctionalization, of members of the DCT family, for the transport of malate into the bundle sheath plastids, occurred during the process of independent recurrent evolution of C 4 photosynthesis in grasses of the PACMAD clade. We also show that this subfunctionalization is lineage independent. Our results challenge the dogma that key C 4 genes must be orthologues of one another among C 4 species, and shed new light on the evolution of C 4 photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum

The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (~0.8 g L –1 ) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L –1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v –1 ) hydrolysates derived from ionic liquid-pretreated sorghum biomass. Further, a key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L –1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol –1 ) from 80 g L –1 glucose and 11.9 g L –1 urea in shake flask batch cultivation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Greater aperture counteracts effects of reduced stomatal density on WUE: a case study on sugarcane and meta-analysis

Stomata regulate CO 2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (g sw ). However, the quantitative relationship between reduced SD, g sw , and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO 2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C 3 dicots, C 3 monocots, and C 4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r 2 =0.60-0.98), but this response is damped relative to the change in anatomy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Meeting liquid biofuel and bioproduct goals: biotechnological design of the intermediate oilseeds pennycress and camelina, and beyond

The European Union and the United States have set ambitious goals to produce biofuels as part of broader decarbonization and energy security initiatives. One of the more feasible routes to liquid biofuels production is the conversion of seed oils [triacylglycerols (TAGs)] to renewable diesel, biodiesel, and sustainable aviation fuel (SAF) using the hydrotreated ester and fatty acids (HEFA) process. Camelina and pennycress are attractive oilseed feedstocks in that they can be grown in the offseason as intermediate crops on tens of millions of hectares of farmland annually, providing ecosystem benefits and not competing with established food crops. Considerably more TAG could be produced by engineering vegetative portions of crops such as sorghum and miscanthus to accumulate economically-viable amounts. Here, this review highlights recent advances in developing pennycress and camelina as intermediate oilseed crops not only for biofuels production but for making higher value oils such as those enriched in astaxanthin, vitamin E, and medium-chain fatty acids. Given the magnitude of renewable liquid fuel demands, we also describe how advances in oil production from vegetative parts of biomass crops can complement intermediate oilseed cropping systems to meet biofuel and bioproduct targets.

biomass crops↗
DOE OSTI2020

No evidence for transient transformation via pollen magnetofection in several monocot species

The development of rapid and efficient transformation methods for many plant species remains an obstacle in both the basic and applied plant sciences. A novel method described by Zhao et al. (2017) used magnetic nanoparticles to deliver DNA into pollen grains of several dicot species, and one monocot (lily), to achieve transformation (“pollen magnetofection”). Using the published protocol, extensive trials by two independent research groups showed no indication of transient transformation success with pollen from two monocots, maize and sorghum. To further address the feasibility of magnetofection, lily pollen was used for side-by-side trials of magnetofection with a proven methodology for transient transformation, biolistics. Using a Green Fluorescent Protein reporter plasmid, transformation efficiency with the biolistic approach averaged 0.7% over three trials. However, the same plasmid produced no recognizable transformants via magnetofection, despite screening >3500 individual pollen grains. We conclude that pollen magnetofection is not effective for transient transformation of pollen for at least three species of monocots, and suggest that efforts to replicate the magnetofection protocol in dicot species would be useful to fully assess its potential. ARISING FROM Zhao et al. Nature Plants https://doi.org/10.1038/s41477-017-0063-z (2017)

Vejlupkova, Zuzana↗
DOE OSTI2020

Associative nitrogen fixation linked with three perennial bioenergy grasses in field and greenhouse experiments

Abstract Associative nitrogen (N 2 )‐fixation (ANF) by bacteria in the root‐zone of perennial bioenergy grasses has the potential to replace or supplement N fertilizer and support sustainable production of biomass, but its application in marginal ecosystems requires further evaluation. In this study, we first combined both greenhouse and field experiments, to explore the N 2 fixation effects of three temperate feedstocks Miscanthus × giganteus (giant miscanthus, Freedom), Panicum virgatum (switchgrass, Alamo), and Saccharum sp. (energycane, Ho 02‐147). In field studies across three growing seasons, plant and soil pools of candidate feedstocks were partially composed of N derived from the atmosphere (Ndfa). Energycane, giant miscanthus, and switchgrass were estimated to derive >30%, %Ndfa. Greenhouse studies were also performed to trace isotopically labeled 15 N 2 into plant biomass and soil pools. Evidence for Ndfa was detected in all three feedstock grasses (using reference 15 N of soil, chicory, and sorghum, δ 15 N~+7.0). Isotopically labeled 15 N 2 was traced into biomass (during grass elongation stage) and soil pools. Extrapolation of rates during the 24 hr labeling period to 50 days estimated 30%–55% of plant Ndfa, with the greatest Ndfa for energycane. The findings of the field natural abundance and greenhouse 15 N 2 feeding experiments provided complementary evidence that perennial bioenergy grasses have the potential to support relatively high rates of ANF, and accumulate diazotroph‐derived N into biomass when grown on non‐fertilized soil.

09 BIOMASS FUELS↗
DOE OSTI2021

Development and validation of time‐domain 1 H‐NMR relaxometry correlation for high‐throughput phenotyping method for lipid contents of lignocellulosic feedstocks

Abstract The bioenergy crops such as energycane, miscanthus, and sorghum are being genetically modified using state of the art synthetic biotechnology techniques to accumulate energy‐rich molecules such as triacylglycerides (TAGs) in their vegetative cells to enhance their utility for biofuel production. During the initial genetic developmental phase, many hundreds of transgenic phenotypes are produced. The efficiency of the production pipeline requires early and minimally destructive determination of oil content in individuals. Current screening methods require time‐intensive sample preparation and extraction with chemical solvents for each plant tissue. A rapid screen will also be needed for developing industrial extraction as these crops become available. In the present study, we have devised a proton relaxation nuclear magnetic resonance ( 1 H‐NMR) method for single‐step, non‐invasive, and chemical‐free characterization of in‐situ lipids in untreated and pretreated lignocellulosic biomass. The systematic evaluation of NMR relaxation time distribution provided insight into the proton environment associated with the lipids in the biomass. It resolved two distinct lipid‐associated subpopulations of proton nuclei that characterize total in‐situ lipids into bound and free oil based on their “molecular tumbling” rate. The T1T2 correlation spectra also facilitated the resolution of the influence of various pretreatment procedures on the chemical composition of molecular and local 1 H population in each sample. Furthermore, we show that hydrothermally pretreated biomass is suitable for direct NMR analysis unlike dilute acid and alkaline pretreated biomass which needs an additional step for neutralization.

pretreatment↗
DOE OSTI2021

Identification and analysis of stem‐specific promoters from sugarcane and energy cane for oil accumulation in their stems

Abstract Considerable recent progress has been achieved in bioengineering oil accumulation in the vegetative tissues of plants, opening an opportunity for large scale production of biodiesel, jet fuel, lubricants, and high‐value lipid bioproducts. For the highly productive C4 crops, such as sugarcane, energy cane, Miscanthus, and fiber sorghums, the bulk of the biomass is the stem. However, little success has been made in accumulating oil in the stem. Since engineering a trait with a constitutive promoter often results in pleiotropic effects that counter trait improvement, identification of stem parenchyma‐specific promoters is a prerequisite for efficient use of the ample photoassimilates stored in mature stem parenchyma cells. In this study, we first identified two TST genes encoding homologues of tonoplast sugar transporters that were strongly and almost exclusively expressed in the stems of canes via a combination of RNA‐seq atlas analysis, in silico analysis of a sugarcane genome, phylogenetic analysis, and quantitative PCR analysis. They were further confirmed in the pith parenchyma cells of the mature stem by RNA in situ hybridization. When fused with the β‐Glucuronidase (GUS) reporter gene, the promoters of two alleles, TST2b‐1A and TST2b‐1C , from one TST gene demonstrated that they could drive the GUS expression exclusively in the stem in Arabidopsis.

09 BIOMASS FUELS↗
DOE OSTI2022

Emerging wild virus of native grass bioenergy feedstock is well–established in the Midwestern USA and associated with premature stand senescence

The North American native prairie grass Panicum virgatum (switchgrass) is a primary bioenergy feedstock candidate. Its widespread distribution and genetic diversity enable the possibility of developing this perennial grass for high production in a variety of conditions, including on marginal lands. A critical concern in feedstock development and deployment is the risk of novel pathogen emergence. Here we investigate the landscape-scale prevalence and epidemiology of a little-studied North American virus first detected in switchgrass and other grasses in bioenergy trials in the US Midwest. Switchgrass mosaic virus (SwMV, Genus Marafivirus, Family Tymoviridae) is transmitted by leafhoppers and phylogenetically sister to Maize rayado fino virus, a significant pathogen of maize in parts of the Americas. Our goal was to determine whether SwMV is uniquely limited to specific bioenergy trials or well-established and circulating more broadly. We used molecular diagnostics to quantify naturally occurring SwMV infection in leafhoppers and switchgrass in naturalistic stands throughout a large Midwestern landscape, and quantified leafhopper abundances and stand performance. Our analysis revealed that this apparently wild virus is well-established and widespread. Infection was present at nearly all sites, across diverse landscape contexts, with prevalences ranging as high as 33%–60%. Infection appeared to accumulate and persist in stands over time. It was associated with increases in premature stand senescence but not with reductions in stand height. Although wild viruses are believed to evolve benign relationships with their natural hosts, these data suggest that SwMV has potential to impact yield components. Viruses are frequently overlooked in crop development efforts, but represent the majority of emerging plant pathogens. For SwMV, it is imperative to quantify its impact on host performance, to identify the extent of any host resistance, and to assess any risks of virus spillover to agricultural plantings of other Poaceae species, including maize and sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Genetics of Flooding Tolerance in an F 2 Miscanthus sacchariflorus ssp. lutarioriparius × M. sinensis Population

Miscanthus is a warm-season, perennial grass cultivated as a feedstock for bioenergy and bioproducts. M. sacchariflorus ssp. lutarioriparius has high yield potential and is well-adapted to seasonal flooding, but little is known about the genetics of this adaptation. We conducted a quantitative trait locus (QTL) analysis on a population of 332 diploid Miscanthus ×giganteus (Mxg) F2s derived from an initial cross between diploid M. sacchariflorus ssp. lutarioriparius ‘PF30022’ and diploid M. sinensis ‘PMS-014’, followed by intermating 50 F 1 s. Using tanks in a greenhouse to assess the effects of partial submergence on actively growing plants, we compared an aerobic soil control to a 6-week flood treatment. The study's primary objectives were to (1) identify QTL for flooding tolerance in Miscanthus , (2) identify candidate genes and (3) compare ethylene response factors in Miscanthus with those in rice and Arabidopsis , sorghum and maize for binding site sequence homology and synteny, especially those associated with flooding tolerance. In total, 10 QTL and 66 candidate genes for partial submergence tolerance were identified (including many for ethylene signalling), a first report for Miscanthus . Notably, none of the Miscanthus candidates were orthologs of rice Sub1A, SK1 or SK2 , yet the ‘PF30022’ parent exhibited a snorkeling phenotype, indicating convergent evolution. This study will facilitate breeding of climate-resiliant Mxg.

abiotic stress tolerance↗
DOE OSTI2026

Contrasting Carbon–Water–Energy Dynamics in Perennial and Annual Bioenergy Agroecosystems Using Eddy Covariance and Interpretable Machine Learning

Understanding how agroecosystems respond to environmental variability is fundamental to predicting productivity and sustainability under a changing climate. We analyzed 55 site-years of high-frequency eddy covariance observations from five agroecosystems—two perennial grasses (miscanthus and switchgrass), two annual rotation systems (maize–soybean and sorghum–soybean), and a restored native prairie—to examine ecosystem-scale carbon, water, and energy fluxes. Using an interpretable machine-learning framework with regression tree ensembles, Shapley Additive Explanations, and Accumulated Local Effects, we quantified how environmental and temporal factors regulate gross primary productivity (GPP), evapotranspiration (ET), water-use efficiency, and the Bowen ratio. Perennials exhibited stronger physiological buffering and maintained fluxes across a broader range of temperature and moisture conditions, reflecting deeper rooting and persistent canopy cover. Annuals, in contrast, showed greater short-term variability and stronger coupling to atmospheric demand, with GPP and ET declining rapidly under low humidity or soil moisture. Differences in temperature sensitivity of Bowen ratio further revealed that perennials sustained proportionally greater sensible heat flux under cool conditions, whereas annuals exhibited constrained energy exchange when evaporative demand was low. Together, these results demonstrate that crop life cycle and canopy structure are fundamental determinants of ecosystem-scale carbon–water–energy coupling. By integrating long-term flux observations with interpretable machine learning, this study identifies the environmental drivers that shape agroecosystem function and highlights how conversion from annual to perennial feedstocks can enhance climatic resilience and alter land–atmosphere energy feedbacks. These findings provide a data-driven basis for improving crop and Earth-system models and for guiding bioenergy landscape design under future climate scenarios.

Accumulated Local Effects↗