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All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Small Signaling Peptides in Sorghum bicolor : Integrating Phylogeny and Gene Expression to Characterize Roles in Stem Development

Small signaling peptides (SSPs) are important regulators of plant growth, development, and responses to biotic and abiotic stress, yet their role in the C4 grass Sorghum bicolor is largely uncharacterized. To help fill this knowledge gap, 219 sorghum genes that encode SSPs were identified based on SSP sequences previously identified in Arabidopsis thaliana, Zea mays, Oryza sativa, Triticum aestivum , and Brachypodium distachyon . The 219 sorghum SSP-encoding genes were assigned to 19 gene families, analyzed for the presence of motifs, and aligned with genes that encode SSPs in other plants using phylogenetic analysis. Sorghum genes in 12 of the 19 SSP gene families had not been previously characterized. Expression of the 219 SSP-encoding genes in sorghum organs, during stem development, and in stem tissues and cell types revealed distinct spatial, temporal, and developmental patterns of expression. Genes associated with the SbCEP and SbRGF families were preferentially expressed in roots, whereas SbEPF genes were expressed in stem epidermal and pith parenchyma cells and panicles. The expression of genes during bioenergy sorghum stem growth and development was investigated because stems account for ~80% of harvested biomass and serve as conduits for water and nutrient transport between leaves and roots. During stem development, 28 SSP genes in several families ( CLE, EPF, CEP, GASS, PSY, ES, PSK, CAPE, POE ) were expressed at higher levels in zones of cell proliferation. For example, the TDIF homologs SbCLE41 and SbCLE42 were expressed at high levels in nascent stem nodes where they may regulate vascular bundle cambial activity and cell differentiation. A different set of 15 genes in the CIF, POE, CAPE, PSY, CEP, RALF , and CLE families were expressed at higher levels in zones of stem tissue differentiation highlighted by elevated expression of five SbRALFR s in the stem nodal plexus. Cell type–specific expression of many sorghum genes that encode SSPs was observed in fully elongated internodes indicating gene expression is regulated with high spatial resolution. Overall, the results provide a foundation of information for analysis of SSP function in sorghum that can be integrated with knowledge of sorghum gene regulatory networks to modulate traits important for production of sorghum crops.

bioenergy sorghum↗
DOE OSTI2021

Epigenetic Control of Drought Response in Sorghum (EPICON)

Genetic manipulation of crops to increase the presence of desirable traits has been critical to increasing agricultural productivity. These changes have primarily involved modification of the plant’s DNA sequence. However, there is increasing evidence that environmental responses are also mediated by epigenetics, which involves heritable changes without changes in DNA sequence. Epigenetic changes have been shown to play a major role in regulating plant responses to drought, an increasing problem worldwide due to climate change. In general, exposure of plants to water limitation triggers epigenetic changes, which include remodeling of chromatin, the network of DNA, RNA and various proteins making up chromosomes, and related changes in regulatory mechanisms. EPICON’s efforts focus on unraveling the role epigenetic signals play in acclimation to and recovery from drought through effects on individual transcription factors or transcriptional networks that direct entire metabolic pathways. To achieve this goal we will follow responses to water deprivation in sorghum, a widely cultivated cereal with recognized drought tolerance. In EPICON’s field trials, sorghum will be grown under controlled irrigation conditions. Leaf and root samples will be taken to perform molecular phenotyping to track changes in epigenetic, transcriptomic, metabolomic and proteomic footprints. Analysis of this data will provide a better understanding of the epigenetic processes related to drought tolerance, leading to our ultimate goal of identifying transcriptional regulators and pathways controlling drought resistance. The identified genetic targets and their regulatory pathways will be used in future efforts to improve growth of sorghum and other crops in the field and in marginal lands under water-limiting conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Development of an open-source regional data assimilation system in PEcAn v. 1.7.2: application to carbon cycle reanalysis across the contiguous US using SIPNET

Abstract. The ability to monitor, understand, and predict the dynamics of the terrestrial carbon cycle requires the capacity to robustly and coherently synthesize multiple streams of information that each provide partial information about different pools and fluxes. In this study, we introduce a new terrestrial carbon cycle data assimilation system, built on the PEcAn model–data eco-informatics system, and its application for the development of a proof-of-concept carbon “reanalysis” product that harmonizes carbon pools (leaf, wood, soil) and fluxes (GPP, Ra, Rh, NEE) across the contiguous United States from 1986–2019. We first calibrated this system against plant trait and flux tower net ecosystem exchange (NEE) using a novel emulated hierarchical Bayesian approach. Next, we extended the Tobit–Wishart ensemble filter (TWEnF) state data assimilation (SDA) framework, a generalization of the common ensemble Kalman filter which accounts for censored data and provides a fully Bayesian estimate of model process error, to a regional-scale system with a calibrated localization. Combined with additional workflows for propagating parameter, initial condition, and driver uncertainty, this represents the most complete and robust uncertainty accounting available for terrestrial carbon models. Our initial reanalysis was run on an irregular grid of ∼ 500 points selected using a stratified sampling method to efficiently capture environmental heterogeneity. Remotely sensed observations of aboveground biomass (Landsat LandTrendr) and leaf area index (LAI) (MODIS MOD15) were sequentially assimilated into the SIPNET model. Reanalysis soil carbon, which was indirectly constrained based on modeled covariances, showed general agreement with SoilGrids, an independent soil carbon data product. Reanalysis NEE, which was constrained based on posterior ensemble weights, also showed good agreement with eddy flux tower NEE and reduced root mean square error (RMSE) compared to the calibrated forecast. Ultimately, PEcAn's new open-source regional data assimilation framework provides a scalable workflow for harmonizing multiple data constraints and providing a uniform synthetic platform for carbon monitoring, reporting, and verification (MRV) as well as accelerating terrestrial carbon cycle research.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Stability of tropical forest tree carbon-water relations in a rainfall exclusion treatment through shifts in effective water uptake depth

Increasing severity and frequency of drought is predicted for large portions of the terrestrial biosphere, with major impacts already documented in wet tropical forests. Using a 4-year rainfall exclusion experiment in the Daintree Rainforest in northeast Australia, we examined canopy tree responses to reduced precipitation and soil water availability by quantifying seasonal changes in plant hydraulic and carbon traits for 11 tree species between control and drought treatments. Even with reduced soil volumetric water content in the upper 1 m of soil in the drought treatment, we found no significant difference between treatments for predawn and midday leaf water potential, photosynthesis, stomatal conductance, foliar stable carbon isotope composition, leaf mass per area, turgor loss point, xylem vessel anatomy, or leaf and stem nonstructural carbohydrates. While empirical measurements of aboveground traits revealed homeostatic maintenance of plant water status and traits in response to reduced soil moisture, modeled belowground dynamics revealed that trees in the drought treatment shifted the depth from which water was acquired to deeper soil layers. Finally, these findings reveal that belowground acclimation of tree water uptake depth may buffer tropical rainforests from more severe droughts that may arise in future with climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

CHESS 2025: Waveform LiDAR data from NEON AOP surveys

This dataset provides Level 1 (L1) full-waveform light detection and ranging (LiDAR) data collected for the 2025 Colorado Headwaters Ecological Spectroscopy Study (CHESS). These data were acquired to enable characterization of vegetation structure and other three-dimensional features of the land surface, and to evaluate structural changes that may have occurred between a prior LiDAR acquisition in 2018 and the 2025 overflight. Waveform LiDAR data can provide more detailed information about objects on the ground than discrete point clouds typically do, and they are often used for granular target segmentation and characterization of subcanopy vegetation. The data were acquired over three study domains in the Upper Gunnison river basin: the upper East River watershed (CRBU); Almont Triangle and Taylor Canyon (ALMO); and Upper Taylor River watershed (UPTA) between 2025-06-13 and 2025-07-15. LiDAR data were acquired using the Optech Galaxy Prime Airborne LiDAR Terrain Mapper onboard the National Ecological Observatory Network (NEON) Airborne Observation Platform (AOP). These are the primary waveform LiDAR data delivered by NEON and are provided per flightline in compressed Pulsewaves format, an open-source binary file standard. A Pulsewaves object comprises a two files: a pulse (.pls) file, which stores the geographic origin, outgoing vector, and metadata for every laser pulse emitted by the scanner, and a wave file (.wvs), which stores the sequential amplitude samples of the outgoing pulse and the returning signals. The files are published here in their compressed forms (.plz, .wvz). All waveform data were processed following the theoretical workflow described in the NEON L0-to-L1 Waveform LiDAR Algorithm Theoretical Basis Document (Krause and Goulden 2022a); however, the Pulsewaves output format differs from a legacy format described in that document. Waveform amplitude samples are recorded at 1 nanosecond intervals. All coordinates are provided in meters. Horizontal coordinates are referenced in Universal Transverse Mercator (UTM) zone 13N and the World Geodetic System (WGS) 1984 ensemble datum. Elevations are referenced to Geoid12A. Waveform data for the UPTA survey area were collected without incident and the published records are complete. However, both the ALMO and CRBU collections experienced issues that resulted in incomplete data for those areas. On collection day 2018-06-16 a hardware failure caused the waveform digitizer to lose data from the eastern edge of the ALMO site (Figure 22). The waveform data for flightlines 2–20 could not be extracted from the digitizer, and the data proved unrecoverable. As a result, a portion of the site does not have coverage with waveform data. Although no hardware failure was observed during collection over the CRBU area, final waveform files generated by vendor software contained only ~25% of the expected number of return pulses. After discovery, NEON initiated troubleshooting with the vendor. The root cause of the data ablation had not been identified at the time of publication. Additional data will be published in an update to this package if further recovery proves successful. CHESS Project Description: The Colorado Headwaters Ecological Spectroscopy Study (CHESS) comprised a multi-week airborne remote sensing and field observation campaign in the Upper Gunnison Basin, Colorado, conducted in June and July of 2025. Airborne remote sensing was conducted by the National Ecological Observatory Network Airborne Observation Platform (NEON AOP), concurrent with a field campaign run by the Rocky Mountain Biological Laboratory (RMBL), the Lawrence Berkeley National Laboratory (LBNL) and SLAC National Accelerator Laboratory Watershed Function Science Focus Area (SFA), and NASA-JPL (Jet Propulsion Laboratory) Earth Surface Mineral Dust Source Investigation (EMIT) program. Between June 10 and July 18, 2025, the NEON AOP flight team collected high-resolution aerial imaging spectroscopy and Light Detection and Ranging (LiDAR) data over three domains: the Upper East River (CRBU), Almont Triangle (ALMO), and the Upper Taylor Basin (UPTA). In coordination with the flights, a field campaign acquired ground-truth observations, including observations of vegetation composition, foliar traits, forest demography, and subsurface properties in 18 core sampling areas within the domains. Additional surface water observations were taken at over 380 point locations. All CHESS campaign datasets can be found within the CHESS ESS-DIVE data portal: https://data.ess-dive.lbl.gov/portals/chess. Funding Acknowledgement: Field and remote-sensing data acquisition was performed under a grant from the National Aeronautics and Space Administration (80NSSC24K1005). This work was also supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

2018 NEON and 2025 CHESS Campaigns↗
DOE OSTI2025

Rhizosphere Microbiome Diversity Potentially Supports Robust Nature of Field Pennycress ( Thlaspi arvense L.) in Dryland Cropping Systems of Eastern Washington

ABSTRACT Field pennycress ( Thlaspi arvense L.) is an annual in the Brassicaceae family and is currently being developed as an oilseed intermediate crop suitable for renewable biodiesel and jet fuel. It displays many desirable characteristics for this role including cold tolerance, a rapid life cycle, and a seed fatty acid profile conducive to bioenergy generation. These traits make field pennycress favorable for winter oilseed cultivation in the inland Pacific Northwest (iPNW). Simultaneously, intermediate crops are an increasingly recognized component of both agronomic sustainability and soil health management. Intermediate crops enhance soil microbial diversity, which benefits both soil and plant health. To understand the impact of field pennycress on soil microbial diversity, two natural accessions and seven experimental accessions were grown at three sites in Eastern Washington. Aboveground biomass and rhizosphere soil were then collected. Soil genomic DNA was extracted from rhizosphere samples and used to generate an amplicon library for bacterial (16S) and fungal (ITS) rRNA sequences. The resulting libraries were analyzed in QIIME2, which revealed that not only did the fad2 deficient line from the Spring32‐10 background have significantly increased aboveground biomass production compared to other pennycress genotypes, but also displayed significantly higher β‐diversity in the rhizosphere community specifically at the site experiencing the driest conditions. ANCOM analysis showed that multiple sequences similar to beneficial plant and soil health enhancing organisms such as Trichoderma spirale , Pseudomonas spp., and Methylobacterium goesingense were found to be enriched in the microbiome of the fad2 Spring32‐10 background also at that site. To add additional context to rhizosphere community data, root exudates from two pennycress genotypes were captured in magenta boxes and analyzed using HPLC. Future work will expand our understanding of the mechanisms by which field pennycress creates diversity in the rhizosphere, thus expanding our ability to cultivate this crop in the iPNW.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Overcoming environmental constraints to high-yielding eastern cottonwood productivity in the southeastern United States

The global demand for biomass is rising, and Populus species are among the most productive temperate woody plants. This 6-year study, conducted in Sumter County, South Carolina, USA evaluated the effects of supplemental irrigation and fertilization on crown and leaf traits, dry weight productivity, and clonal variation in Populus deltoides. Eight commercially available clones (‘ST148’, ‘S2C20’, ‘3167’, ‘ST72’, ‘20WV’, ‘S7C1’, ‘ST70’, and ‘S7C15’) were assessed. Drip irrigation was applied to maintain soil tension at either 33 kPa (15% volumetric soil moisture) or 1500 kPa (10%). Nutrient treatments via weekly fertigation applications consisted of two levels: a high annual rate (initially 85 kg elemental nitrogen ha−1 year−1, increasing to 182 kg ha−1 year−1 at Year 3, then reduced to 135 kg ha−1 year−1 thereafter) and a low rate at 50% of the high treatment. Dry weight yields were estimated using standing tree measurements. Results revealed significant clonal differences in leaf area index (LAI) that peaked in Year 3, with the fastest-growing clones exhibiting lower LAI values. At Ages 2 and 3, these clones also showed higher concentrations of K, Ca, B, Cu, and Mn in leaves, along with lower Mg and Zn concentrations. Both annual height and diameter growth varied significantly by treatment and clone, with the largest height increases observed in the low fertilizer treatments from Year 3 onward. Biomass production in all irrigated plots exceeded that of non-irrigated plots across all biomass components at Ages 4, 5, and 6. Furthermore, stem-to-root dry weight ratios increased more rapidly in the best-performing treatment–clone combinations compared to non-irrigated plots. Notably, P. deltoides plots receiving fertilizer had higher soil carbon concentrations (i.e., percent C) compared to adjacent sweetgum, sycamore, and agricultural plots. These findings highlight the potential of targeted irrigation strategies to enhance biomass productivity in P. deltoides plantations.

Tuskan, Gerald [ORNL] (ORCID:0000000301061289)↗
DOE OSTI2021

Tree Canopies Reflect Mycorrhizal Composition

Mycorrhizae alter global patterns of CO 2 fertilization, carbon storage, and elemental cycling, yet knowledge of their global distributions is currently limited by the availability of forest inventory data. Here, we show that maps of tree-mycorrhizal associations (hereafter “mycorrhizal maps”) can be improved by the novel technology of imaging spectroscopy because mycorrhizal signatures propagate up from plant roots to impact forest canopy chemistry. We analyzed measurements from 143 airborne imaging spectroscopy surveys over 112,975 individual trees collected across 13 years. Results show remarkable accuracy in capturing ground truth observations of mycorrhizal associations from canopy signals across disparate landscapes (R 2 = 0.92, p < 0.01). Additionally, upcoming imaging spectroscopy satellite missions can reveal new insights into landscape-scale variations in water, nitrogen, phosphorus, carotenoid/anthocyanin, and cellulose/lignin composition. Applied globally, this approach could improve the spatial precision of mycorrhizal distributions by a factor of roughly 104 and facilitate the incorporation of dynamic shifts in forest composition into Earth system models.

58 GEOSCIENCES↗
DOE OSTI2025

Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping

Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. Furthermore, these innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Plant Characteristics, Porewater, Gas Flux, and Soil Biogeochemistry at Council Road Site Mile Marker 71, Seward Peninsula, Alaska, 2023

Data collected at Council, AK (64°51’35.0”N 163°41’59.1”W) during a summer campaign in 2023. Water data consists of soil porewater collected by centrifuging soil cores and also by field collection with porewater samplers (rhizons). Gas data consists of CO2, CH4 and N2O surface soil fluxes measured with a portable FTIR analyzer. Plant and root data consists of biomass, root length, diameter and mass. Soil data consists of total C and N. Air, water and soil samples span two main locations: a thermokarst wetland and an upland tussock. The Next-Generation Ecosystem Experiments: Arctic (NGEE Arctic), was a research effort to reduce uncertainty in Earth System Models by developing a predictive understanding of carbon-rich Arctic ecosystems and feedbacks to climate. NGEE Arctic was supported by the Department of Energy's Office of Biological and Environmental Research.The NGEE Arctic project had two field research sites: 1) located within the Arctic polygonal tundra coastal region on the Barrow Environmental Observatory (BEO) and the North Slope near Utqiagvik (Barrow), Alaska and 2) multiple areas on the discontinuous permafrost region of the Seward Peninsula north of Nome, Alaska. Through observations, experiments, and synthesis with existing datasets, NGEE Arctic provided an enhanced knowledge base for multi-scale modeling and contributed to improved process representation at global pan-Arctic scales within the Department of Energy's Earth system Model (the Energy Exascale Earth System Model, or E3SM), and specifically within the E3SM Land Model component (ELM).This dataset was generated to broadly address the following research question: how will climate change (i.e., thawing permafrost, landscape change) alter the ecosystem flux (sink versus source) of important greenhouse gases such as CO2, CH4 and N2O?Description of the contents of this data package: This dataset contains 5 different individual .csv files containing plant, soil, water and gas data. No software is needed to utilize them. PFTCover: Plant functional type ground cover in 1x1 meter plots. SoilCores: Solidphase and porewater phase soil biogeochemical variablesPlantData: Above and belowground plant traits. GasFlux: Surface plant-soil gas measurementsFieldPorewater: Field collected porewater biogeochemical variables

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Data for "Genetics of flooding tolerance in an F2 Miscanthus sacchariflorus ssp. lutarioriparius × M. sinensis population"

This dataset contains all data and supplementary materials from "Genetics of flooding tolerance in an F2 Miscanthus sacchariflorus ssp. lutarioriparius × M. sinensis population". 1. The dataset S1 table contains the raw phenotypic data collected during the experiment. 2. The dataset S2 table contains the LSmean values for the 24 traits studied. 3. The dataset S3 table contains the TASSEL GBSv2 map, marker information, and genotype data used for mapping. 4. The dataset S4 table contains information on candidate genes found in each of the QTL intervals. 5. The dataset S5 table contains the GO annotations and KEGG enrichment analyses for those candidate genes. 6. The dataset S6 table contains information on the sequences used to classify AP2 ERF transcription factors. 7. The dataset S7 table contains information on AP2 ERF orthologs between Miscanthus and rice based on synteny. 8. Supplementary file 1 contains the ANOVA results using the raw phenotypic data collected from protocol "A". 9. Supplementary file 2 contains the ANOVA results using the raw phenotypic data collected from protocol "B". 10. Supplementary file 3 contains notes on the comparison of SNP calling methods. 11. Supplementary file 4 is a script for analyzing candidate genes found in QTL intervals.

Miscanthus, flood, partial submergence, complete s↗
DOE OSTI2022

HT-SIP: a semi-automated stable isotope probing pipeline identifies cross-kingdom interactions in the hyphosphere of arbuscular mycorrhizal fungi

Abstract Background Linking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive for this goal, Stable-isotope probing—SIP—remains among the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, actively growing microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated, high-throughput SIP (HT-SIP) pipeline to support well-replicated, temporally resolved amplicon and metagenomics experiments. We applied this pipeline to a soil microhabitat with significant ecological importance—the hyphosphere zone surrounding arbuscular mycorrhizal fungal (AMF) hyphae. AMF form symbiotic relationships with most plant species and play key roles in terrestrial nutrient and carbon cycling. Results Our HT-SIP pipeline for fractionation, cleanup, and nucleic acid quantification of density gradients requires one-sixth of the hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We applied HT-SIP to 13 C-AMF hyphosphere DNA from a 13 CO 2 plant labeling study and created metagenome-assembled genomes (MAGs) using high-resolution SIP metagenomics (14 metagenomes per gradient). SIP confirmed the AMF Rhizophagus intraradices and associated MAGs were highly enriched (10–33 atom% 13 C), even though the soils’ overall enrichment was low (1.8 atom% 13 C). We assembled 212 13 C-hyphosphere MAGs; the hyphosphere taxa that assimilated the most AMF-derived 13 C were from the phyla Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia-oxidizing archaeon genus Nitrososphaera . Conclusions Our semi-automated HT-SIP approach decreases operator time and improves reproducibility by targeting the most labor-intensive steps of SIP—fraction collection and cleanup. We illustrate this approach in a unique and understudied soil microhabitat—generating MAGs of actively growing microbes living in the AMF hyphosphere (without plant roots). The MAGs’ phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Data for "Enhancing Lipid Production in Plant Cells through Automated High-Throughput Genome Engineering and Phenotyping"

Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

AI/ML↗
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↗