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149 records · Page 9

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DOE OSTI2023

A temporal analysis and response to nitrate availability of 3D root system architecture in diverse pennycress (Thlaspi arvense L.) accessions

Introduction Roots have a central role in plant resource capture and are the interface between the plant and the soil that affect multiple ecosystem processes. Field pennycress ( Thlaspi arvense L.) is a diploid annual cover crop species that has potential utility for reducing soil erosion and nutrient losses; and has rich seeds (30-35% oil) amenable to biofuel production and as a protein animal feed. The objective of this research was to (1) precisely characterize root system architecture and development, (2) understand plastic responses of pennycress roots to nitrate nutrition, (3) and determine genotypic variance available in root development and nitrate plasticity. Methods Using a root imaging and analysis pipeline, the 4D architecture of the pennycress root system was characterized under four nitrate regimes, ranging from zero to high nitrate concentrations. These measurements were taken at four time points (days 5, 9, 13, and 17 after sowing). Results Significant nitrate condition response and genotype interactions were identified for many root traits, with the greatest impact observed on lateral root traits. In trace nitrate conditions, a greater lateral root count, length, density, and a steeper lateral root angle was observed compared to high nitrate conditions. Additionally, genotype-by-nitrate condition interaction was observed for root width, width:depth ratio, mean lateral root length, and lateral root density. Discussion These findings illustrate root trait variance among pennycress accessions. These traits could serve as targets for breeding programs aimed at developing improved cover crops that are responsive to nitrate, leading to enhanced productivity, resilience, and ecosystem service.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

RhizoVision Explorer: open-source software for root image analysis and measurement standardization

Abstract Roots are central to the function of natural and agricultural ecosystems by driving plant acquisition of soil resources and influencing the carbon cycle. Root characteristics like length, diameter and volume are critical to measure to understand plant and soil functions. RhizoVision Explorer is an open-source software designed to enable researchers interested in roots by providing an easy-to-use interface, fast image processing and reliable measurements. The default broken roots mode is intended for roots sampled from pots and soil cores, washed and typically scanned on a flatbed scanner, and provides measurements like length, diameter and volume. The optional whole root mode for complete root systems or root crowns provides additional measurements such as angles, root depth and convex hull. Both modes support providing measurements grouped by defined diameter ranges, the inclusion of multiple regions of interest and batch analysis. RhizoVision Explorer was successfully validated against ground truth data using a new copper wire image set. In comparison, the current reference software, the commercial WinRhizo™, drastically underestimated volume when wires of different diameters were in the same image. Additionally, measurements were compared with WinRhizo™ and IJ_Rhizo using a simulated root image set, showing general agreement in software measurements, except for root volume. Finally, scanned root image sets acquired in different labs for the crop, herbaceous and tree species were used to compare results from RhizoVision Explorer with WinRhizo™. The two software showed general agreement, except that WinRhizo™ substantially underestimated root volume relative to RhizoVision Explorer. In the current context of rapidly growing interest in root science, RhizoVision Explorer intends to become a reference software, improve the overall accuracy and replicability of root trait measurements and provide a foundation for collaborative improvement and reliable access to all.

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 OSTI2022

Root system architecture and environmental flux analysis in mature crops using 3D root mesocosms

Current methods of root sampling typically only obtain small or incomplete sections of root systems and do not capture their true complexity. To facilitate the visualization and analysis of full-sized plant root systems in 3-dimensions, we developed customized mesocosm growth containers. While highly scalable, the design presented here uses an internal volume of 45 ft 3 (1.27 m 3 ), suitable for large crop and bioenergy grass root systems to grow largely unconstrained. Furthermore, they allow for the excavation and preservation of 3-dimensional root system architecture (RSA), and facilitate the collection of time-resolved subterranean environmental data. Sensor arrays monitoring matric potential, temperature and CO 2 levels are buried in a grid formation at various depths to assess environmental fluxes at regular intervals. Methods of 3D data visualization of fluxes were developed to allow for comparison with root system architectural traits. Following harvest, the recovered root system can be digitally reconstructed in 3D through photogrammetry, which is an inexpensive method requiring only an appropriate studio space and a digital camera. We developed a pipeline to extract features from the 3D point clouds, or from derived skeletons that include point cloud voxel number as a proxy for biomass, total root system length, volume, depth, convex hull volume and solidity as a function of depth. Ground-truthing these features with biomass measurements from manually dissected root systems showed a high correlation. We evaluated switchgrass, maize, and sorghum root systems to highlight the capability for species wide comparisons. We focused on two switchgrass ecotypes, upland (VS16) and lowland (WBC3), in identical environments to demonstrate widely different root system architectures that may be indicative of core differences in their rhizoeconomic foraging strategies. Finally, we imposed a strong physiological water stress and manipulated the growth medium to demonstrate whole root system plasticity in response to environmental stimuli. Hence, these new “3D Root Mesocosms” and accompanying computational analysis provides a new paradigm for study of mature crop systems and the environmental fluxes that shape them.

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↗