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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A Novel Gene Stacking Method in Plant Transformation Utilizing Split Selectable Markers

Gene stacking, the process of introducing multiple genes into a single plant to enhance desired traits, is essential for plant genetic improvement through both conventional breeding and genetic transformation. In general, transformation-based gene stacking can be achieved through either co-transformation to simultaneously introduce multiple genes or sequential multi-round transformation. While co-transformation is generally faster and more efficient than sequential multi-round transformation, it often requires two selectable marker genes, which confer resistance to antibiotics, for selecting transgenic events. However, in most cases, there is only one best selectable marker gene for a specific plant species or genotype. Also, it is harder to optimize the concentrations of two antibiotics for co-transformation than using one antibiotic for selecting transgenic events. To overcome this challenge, we recently developed an innovative split selectable marker system for plant co-transformation, allowing the use of one selectable marker gene to select transgenic events. This method involves constructing two binary vectors, each carrying a subset of genes of interest and a partial fragment of the selectable marker gene, which is connected to a partial intein fragment. Following Agrobacterium -mediated co-transformation, plants harboring both binary vectors are selected using a single antibiotic, such as kanamycin. This split-marker system can be used to co-transform multiple genes into both herbaceous and woody plants, accelerating genetic improvement of polygenic traits or integrative improvement of multiple traits to simultaneously increase crop yield and quality.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A genetic link between leaf carbon isotope composition and whole-plant water use efficiency in the C 4 grass Setaria

Genetic selection for whole-plant water use efficiency (yield per transpiration; WUE plant ) in any crop-breeding programme requires high-throughput phenotyping of component traits of WUE plant such as intrinsic water use efficiency (WUE i ; CO 2 assimilation rate per stomatal conductance). Measuring WUE i by gas exchange measurements is laborious and time consuming and may not reflect an integrated WUE i over the life of the leaf. Alternatively, leaf carbon stable isotope composition (δ 13 C leaf ) has been suggested as a potential time-ntegrated proxy for WUE i that may provide a tool to screen for WUE plant . However, a genetic link between δ 13 C leaf and WUE plant in a C 4 species has not been well established. Thus, to confirm if there is a genetic relationship in a C 4 plant between δ 13 C leaf and WUE plant under well watered and water-limited growth conditions, a high-throughput phenotyping facility was used to measure WUE plant in a recombinant inbred line (RIL) population created between the C 4 grasses Setaria viridis and S. italica . Three quantitative trait loci (QTL) for δ 13 C leaf were found and co-localized with transpiration, biomass accumulation, and WUE plant . Additionally, WUE plant for each of the δ 13 C leaf QTL allele classes was negatively correlated with δ 13 C leaf , as would be predicted when WUE i influences WUE plant . These findings demonstrate that δ 13 C leaf is genetically linked to WUE plant , likely to be through their relationship with WUE i , and can be used as a high-throughput proxy to screen for WUE plant in these C 4 species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Protoplast-Based Transient Expression and Gene Editing in Shrub Willow ( Salix purpurea L .)

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Synthetic feed-forward loop circuit boosts transgene expression in sugarcane

Bio-based products derived from natural plant-derived materials offer a promising alternative to petroleum-based products, which are essential for achieving global sustainability (Gupta et al. 2022). Sugarcane provides 40% of the world’s biofuel (Brant et al. 2025). Recently, metabolic engineering of sugarcane for hyperaccumulation of biomass oil is emerging as a strategy to elevate the crop’s energy content (Cao et al. 2023; Maitra et al. 2024). Synthetic transcription factors offer a powerful tool for modulating entire metabolic pathways by enabling fine-tuned activation or repression of specific genes (Liu and Stewart 2016; Hooghvorst and Altpeter 2023). However, the application of these technologies faces challenges, including the availability of well-characterized genetic building blocks for precise manipulation of gene expression, and the inherently challenging properties of gene expression in highly polyploid crops like sugarcane (Liu and Stewart 2016).

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

A large sequenced mutant library – valuable reverse genetic resource that covers 98% of sorghum genes

SUMMARY Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum , a drought and heat‐tolerant C4 species, requires a vast, large‐scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large‐scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)‐induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis ‐regulatory elements. A comparison of the distribution of mutations in the large‐scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS‐induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5′ UTR, gene body, and 3′‐UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the K a / K s value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB ( https://www.depts.ttu.edu/igcast/sorbmutdb.php ) and SorghumBase ( https://sorghumbase.org/ ). This current large‐scale sequence‐indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Rays for Roots - Integrating Backscatter X-Ray Phenotyping, Modeling and Genetics to Increase Carbon Sequestration and Switchgrass Resource Use (Final Report)

To increase carbon (C) deposition in the soil and enhance crop resource use efficiency, characterizing root form and function is essential. Several root and soil traits have been linked to increased root-to-soil C transfer. Technology that could provide high-resolution characterization of many of these traits in field conditions would revolutionize our ability to study and understand how to increase C sequestration. In this effort, we developed an initial early prototype backscatter X-ray system for non-destructive imaging of root traits. We collected initial backscatter X-ray data in field and lab settings and carried out early analysis of these data. Along with this prototype, we also developed a suite of root phenotyping approaches including advanced minirhizotron image analysis, soil core imaging, and mesocosm imaging. Minirhizotron (MR) tubes are clear tubes inserted into the soil in the field and used to image roots and the surrounding soil. Our team has developed deep learning-based methods that can segment roots from soil that can learn from imprecise image-level labels. The ability to learn or fine-tune our deep learning algorithms from image-level labels allows easier and faster application of these approaches to new locations and new plant species. We have successfully implemented and applied our MR analysis approaches to thousands of switchgrass MR images collected across geographical regions. An advantage of MR imaging is the ability to collect root and soil images over time. Our soil core analysis included collecting hundreds of soil core samples from harvested switchgrass fields and imaging these cores with both X-ray CT and backscatter X-ray imaging. Initial segmentation approaches for the X-ray CT images of these cores have been developed and applied. An advantage of soil core analysis is that it preserves the three-dimensional structures of the roots and soil in the core collected. Our group also developed photogrammetry-based mesocosm root imaging and phenotyping approaches. In this approach, a plant was grown in a large mesocosm with a three-dimensional grid of thin supporting lines inserted throughout the mesocosm. After the plant (and, correspondingly, the root architecture is grown and established) the soil media was removed and the supporting lines approximately preserved the three-dimensional root architecture. Then, we applied photogrammetry techniques to create a three-dimensional digital representation of the root architecture for which we developed analysis algorithms including skeletonization. We carried out our phenotyping development with powerful switchgrass resources and physiological and agroecosystem modeling to deliver novel technology. This project contributes to multiple ARPA-E missions including reduction of foreign imports of energy, reduction of energy-related emissions including greenhouse gases, and ensuring that the United States maintains a technological lead in developing and deploying advanced energy technology. Furthermore, the developed tools could transform public and private plant breeding and could be broadly applicable to other crops and, potentially, other application areas. Our team of engineers, plant and soil scientists, and modelers i) developed an early prototype backscatter X-ray platform that can operate in field conditions; ii) developed a suite of root phenotyping and characterization approaches as described above; iii) developed and carried out plant biology and physiology roots studies and; iv) developed and implemented mechanistic physiological modeling.

42 ENGINEERING↗
DOE OSTI2025

A Chromosome-Scale Genome Assembly of the Flax Rust Fungus Reveals the Two Unusually Large Effector Proteins, AvrM3 and AvrN

Rust fungi comprise thousands of species, many of which cause disease on important crop plants. The flax rust fungus Melampsora lini has been a model species for the genetic dissection of plant immunity since the 1940s; however, the highly fragmented and incomplete reference genome has so far hindered progress in effector gene discovery. Here, we generated a fully phased, chromosome-scale assembly of the two nuclear genomes of M. lini strain CH5, resolving an additional 320 Mbp of the sequence. The 482-Mbp dikaryotic genome is at least 79% repetitive, with a large proportion (approximately 40%) of the genome comprising young, highly similar transposable elements. The assembly resolves the known effector gene loci, some of which carry complex duplications that were collapsed in the previous assembly. Using a genetic map followed by manual correction of gene models, we identified the AvrM3 and AvrN genes, which encode unusually large fungal effector proteins and trigger defense responses when co-expressed with the corresponding resistance genes. We located the genes linked to the tetrapolar mating system on chromosomes 4 and 9, but in contrast to the cereal rusts that have one pheromone receptor gene per haplotype, in flax rust, three pheromone receptor genes were found, with two of them closely linked on one haplotype. Taken together, we show that a high-quality assembly is crucial for resolving complex gene loci, and given the increasing number of fungal effectors of large size, the commonly applied criterion for effector candidates of being small proteins needs to be reconsidered.

Melampsora↗
DOE OSTI2023

Metabolic and transcriptomic study of pennycress natural variation identifies targets for oil improvement

Pennycress (Thlaspi arvense L.), a member of the Brassicaceae family, produces seed oil high in erucic acid, suitable for biodiesel and aviation fuel. Although pennycress, a winter annual, could be grown as a dedicated bioenergy crop, an increase in its seed oil content is required to improve its economic competitiveness. The success of crop improvement relies upon finding the right combination of biomarkers and targets, and the best genetic engineering and/or breeding strategies. In this work, we combined biomass composition with metabolomic and transcriptomic studies of developing embryos from 22 pennycress natural variants to identify targets for oil improvement. The selected accession collection presented diverse levels of fatty acids at maturity ranging from 29% to 41%. Pearson correlation analyses, weighted gene co‐expression network analysis and biomarker identifications were used as complementary approaches to detect associations between metabolite level or gene expression and oil content at maturity. The results indicated that improving seed oil content can lead to a concomitant increase in the proportion of erucic acid without affecting the weight of embryos. Processes, such as carbon partitioning towards the chloroplast, lipid metabolism, photosynthesis, and a tight control of nitrogen availability, were found to be key for oil improvement in pennycress. Besides identifying specific targets, our results also provide guidance regarding the best timing for their modification, early or middle maturation. Thus, this work lays out promising strategies, specific for pennycress, to accelerate the successful development of lines with increased seed oil content for biofuel applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

EvoNet: A phylogenomic and systems biology approach to identify genes underlying plant survival in marginal, low‐N soils

The DOE‐BER “EvoNet” project investigates the genetic and molecular basis of plant resilience in extreme environments. We do this by identifying key genes that enable “extreme survivor” species to thrive in the nitrogen-poor soils of Chile’s hyper-arid Atacama Desert. Our collections focus on 32 Atacama extremophile species, including seven grass species with potential biofuel applications. To identify genes-of-importance to survival we compared genomic and transcriptomic profiles of extremophile species that thrive in the Atacama to those of closely related “sister” species from nitrogen-rich arid and mesic regions of California. Deep RNA sequencing and de novo transcriptome assembly across these triplet species sets supported a phylogenomic framework for identifying positively selected genes associated with adaptive divergence. Our integrative analysis combined ecological and environmental data, metagenomics, evolutionary and systems biology, and metabolomics. This enabled us to create an unprecedented framework for systematically understanding how non-model plants have adapted to survive in extreme conditions. Our resulting database of positively selected ortholog groups in the extremophile plants offers promising targets for engineering crop and biofuel species with enhanced resilience to drought and extreme weather. Additionally, our newest dataset explores and exploits a complementary metabolomic approach. This new aspect provides innovative strategies to manipulate plant cell metabolism, further supporting efforts to improve agricultural productivity in the face of extreme climates. Importantly, our combined evolutionary- and metabolomic-based strategies focused on convergent patterns of adaptation, providing a genetic and metabolomic toolkit for improving crop and biofuel resilience across diverse plant species. Finally, our novel exploration of ecological and evolutionary dynamics delivered to the community a phylogenomic computational pipeline called “PhyloGeneious.” Our continued adaptations of this pipeline are publicly available to expedite evolutionary genomic research for future scientific discoveries. In total, our DOE-BER has provided genomic, metabolomic, and computational strategies to understand how extremophile plants provide evolutionary and physiological targets for improving agricultural and biofuel production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Host genetic variation drives the differentiation in the ecological role of the native Miscanthus root-associated microbiome

Microbiome recruitment is influenced by plant host, but how host plant impacts the assembly, functions, and interactions of perennial plant root microbiomes is poorly understood. Here we examined prokaryotic and fungal communities between rhizosphere soils and the root endophytic compartment in two native Miscanthus species (Miscanthus sinensis and Miscanthus floridulus) of Taiwan and further explored the roles of host plant on root-associated microbiomes. Our results suggest that host plant genetic variation, edaphic factors, and site had effects on the root endophytic and rhizosphere soil microbial community compositions in both Miscanthus sinensis and Miscanthus floridulus, with a greater effect of plant genetic variation observed for the root endophytic communities. Host plant genetic variation also exerted a stronger effect on core prokaryotic communities than on non-core prokaryotic communities in each microhabitat of two Miscanthus species. From rhizosphere soils to root endophytes, prokaryotic co-occurrence network stability increased, but fungal co-occurrence network stability decreased. Furthermore, we found root endophytic microbial communities in two Miscanthus species were more strongly driven by deterministic processes rather than stochastic processes. Root-enriched prokaryotic OTUs belong to Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Sphingobacteriia, and [Saprospirae] both in two Miscanthus species, while prokaryotic taxa enriched in the rhizosphere soil are widely distributed among different phyla. We provide empirical evidence that host genetic variation plays important roles in root-associated microbiome in Miscanthus. The results of this study have implications for future bioenergy crop management by providing baseline data to inform translational research to harness the plant microbiome to sustainably increase agriculture productivity.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Time‐series multi‐omics analysis of micronutrient stress in Sorghum bicolor reveals iron and zinc crosstalk and regulatory network conservation

Micronutrient stress impacts growth, biomass production, and grain yield in crops. Multi-omics studies are valuable resources in identifying genes for functional studies and trait improvement, such as accumulation of Fe or Zn under deficient or excess conditions for bioenergy or grain agriculture. We conducted transcriptomics and ionomics analyses on Sorghum bicolor BTx623, grown under Fe and Zn limited and excess conditions over a 21-day period. To identify early and late transcriptional response in roots and leaves, 180 RNAseq libraries were sequenced for differential expression and co-expression network analyses. Fe and Zn accumulation was measured using ICP-MS at each time point, and a fluorometer was used to estimate chlorophyll content in leaves. Among the four treatments, Fe limitation and Zn excess resulted in the largest phenotypic effects and transcriptional response in roots and leaves. Several of the reduction (Strategy I) and chelation (Strategy II) strategy genes that improve bioavailability of Fe and Zn in plant roots often used by non-grass and grass species, respectively, were differentially expressed. Gene regulatory network (GRN) analysis of roots revealed enrichment of genes from Fe limiting and Zn excess which strongly connect to homologues of SbFIT, SbPYE, and SbBTS as hub genes. The GRN for leaf responses showed homologues of SbPYE and SbBTS as hubs connecting genes for chloroplast biosynthesis, Fe-S cluster assembly, photosynthesis, and ROS scavenging. Expression analyses suggest sorghum uses Strategy II genes for Fe and Zn uptake, as expected, but can also utilize Strategy I genes, which may be advantageous in variable moisture environments. We found strong overlap between Fe and Zn responsive GRNs, indicative of micronutrient crosstalk. We also found conservation of root and leaf GRNs, and known homologous genes suggest strong constraints on homeostasis networks in plants. These data will provide a resource for functional genetics to enhance micronutrient transport in sorghum, and opportunities to conduct further comparative GRN analysis across diverse crops species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genetic control of photoprotection and photosystem II operating efficiency in plants

Summary Photoprotection against excess light via nonphotochemical quenching (NPQ) is indispensable for plant survival. However, slow NPQ relaxation under low light conditions can decrease yield of field‐grown crops up to 40%. Using semi‐high‐throughput assay, we quantified the kinetics of NPQ and photosystem II operating efficiency (ΦPSII) in a replicated field trial of more than 700 maize ( Zea mays ) genotypes across 2 yr. Parametrized kinetics data were used to conduct genome‐wide association studies. For six candidate genes involved in NPQ and ΦPSII kinetics in maize the loss of function alleles of orthologous genes in Arabidopsis ( Arabidopsis thaliana ) were characterized: two thioredoxin genes, and genes encoding a transporter in the chloroplast envelope, an initiator of chloroplast movement, a putative regulator of cell elongation and stomatal patterning, and a protein involved in plant energy homeostasis. Since maize and Arabidopsis are distantly related, we propose that genes involved in photoprotection and PSII function are conserved across vascular plants. The genes and naturally occurring functional alleles identified here considerably expand the toolbox to achieving a sustainable increase in crop productivity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparisons of biomass, water use efficiency and water use strategies across five genomic groups of Populus and its hybrids

Abstract Genetic improvement and hybridization in the Populus genus have led to the development of genotypes exhibiting fast growth, high rooting ability and disease resistance. However, while large biomass production is important for bioenergy crops, efficient use of resources including water is also important in sites lacking irrigation and for maintaining ecosystem water availability. In addition, comparison of water use strategies across a range of growth rates and genetic variability can elucidate whether certain strategies are shared among the fastest growing and/or most water use efficient genotypes. We estimated tree water use throughout the second growing season via sapflow sensors of 48 genotypes from five Populus taxa; P. deltoides W. Bartram ex Marshall × P. deltoides (D × D), P. deltoides × P. maximowiczii A. Henry (D × M), P. deltoides × P. nigra L. (D × N), P. deltoides × P. trichocarpa Torr. & Gray (D × T) and P. trichocarpa × P. deltoides (T × D) and calculated average canopy stomatal conductance ( G S ). We regressed G S and atmospheric vapor pressure deficit (VPD) wherein the slope of the relationship represents stomatal sensitivity to VPD. At the end of the second growing season, trees were harvested, and their dry woody biomass was used to calculate whole tree water use efficiency (WUE T ). We found that D × D and D × M genotypes exhibited differing water use strategies with D × D genotypes exhibiting high stomatal sensitivity while retaining leaves while D × M genotypes lost leaf area throughout the growing season but exhibited low stomatal sensitivity. Across measured taxa, biomass growth was positively correlated with WUE T , and genotypes representing each measured taxa except D × N and T × D had high 2‐year dry biomass of above 6 kg/tree. Overall, these data can be used to select Populus genotypes that combine high biomass growth with stomatal sensitivity and WUE T to limit the negative impacts of bioenergy plantations on ecosystem water resources.

09 BIOMASS FUELS↗
DOE OSTI2020

Prospects for Engineering Biophysical CO 2 Concentrating Mechanisms into Land Plants to Enhance Yields

Although cyanobacteria and algae represent a small fraction of the biomass of all primary producers, their photosynthetic activity accounts for roughly half of the daily CO 2 fixation that occurs on Earth. These microorganisms are able to accomplish this feat by enhancing the activity of the CO 2 -fixing enzyme Rubisco using biophysical CO 2 concentrating mechanisms (CCMs). Biophysical CCMs operate by concentrating bicarbonate and converting it into CO 2 in a compartment that houses Rubisco (in contrast with other CCMs that concentrate CO 2 via an organic intermediate, such as malate in the case of C 4 CCMs). This activity provides Rubisco with a high concentration of its substrate, thereby increasing its reaction rate. The genetic engineering of a biophysical CCM into land plants is being pursued as a strategy to increase crop yields. This review focuses on the progress toward understanding the molecular components of cyanobacterial and algal CCMs, as well as recent advances toward engineering these components into land plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A KBase Case Study on Genome-wide Transcriptomics and Plant Primary Metabolism in Sorghum

A better understanding of the genetic and metabolic mechanisms that confer stress resistance and tolerance in plants is key to engineering new crops through advanced breeding technologies. This requires a systems biology approach that builds on a genome-wide understanding of the regulation of gene expression, plant metabolism, physiology and growth. In this study, we examine the response to drought stress in Sorghum, as we leverage the tools for transcriptomics and plant metabolic modeling we have implemented at the U.S. Department of Energy Systems Biology Knowledgebase (KBase). KBase enables researchers worldwide to collaborate and advance research by allowing them to upload private or public data into the KBase Narrative Interface, empowering them to analyze this data using a rich, extensible array of computational and data-analytics tools, and allows them to securely share scientific workflows and conclusions. We demonstrate how to use the current RNA-seq tools in KBase, applicable to both plants and microbes, to assemble and quantify long transcripts and identify differentially expressed genes effectively. More specifically, we demonstrate the utility of the platform by identifying key genes that are differentially expressed during drought-stress in Sorghum bicolor, which is an important sustainable production crop plant. We then show how to use KBase tools to predict the membership of genes in metabolic pathways and examine expression data in the context of metabolic subsystems. We demonstrate the power of the platform by making the data analysis and interpretation available to the biologists in the reproducible, re-usable, point-and-click format of a KBase Narrative thus promoting FAIR (Findable, Accessible, Interoperable and Reusable) guiding principles for scientific data management and stewardship.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Turbo‐charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene‐free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High‐throughput sequencing technologies, including long‐read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements—often missed by standard genotyping—when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user‐friendly, synthetic biology‐compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue‐specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next‐generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A co-opted steroid synthesis gene, maintained in sorghum but not maize, is associated with a divergence in leaf wax chemistry

Significance Virtually all above-ground plant surfaces, such as leaf and stem exteriors, are covered in a cuticle: a wax-infused polyester. This waxy biocomposite is the largest interface between Earth’s biosphere and atmosphere. Its chemical composition is not only highly tuned to mediate nonstomatal water loss, but it also self-assembles to produce superhydrophobic surfaces, protects against UV radiation, and contains bioactive compounds that help resist microbial attack. Developing fundamental knowledge of waxy biocomposites, particularly those on crop species, is a prerequisite for an understanding of their structure–function relationships. Here, we uncover a likely genetic basis for the presence and absence, respectively, of triterpenoids in the leaf waxes of sorghum and maize—compounds previously associated with creating heat-tolerant cuticular water barriers.

09 BIOMASS FUELS↗
DOE OSTI2022

Uncoupling differential water usage from drought resistance in a dwarf Arabidopsis mutant

Abstract Understanding the molecular and physiological mechanisms of how plants respond to drought is paramount to breeding more drought-resistant crops. Certain mutations or allelic variations result in plants with altered water-use requirements. To correctly identify genetic differences which confer a drought phenotype, plants with different genotypes must be subjected to equal levels of drought stress. Many reports of advantageous mutations conferring drought resistance do not control for soil water content (SWC) variations across genotypes and may therefore need to be re-examined. Here, we reassessed the drought phenotype of the Arabidopsis (Arabidopsis thaliana) dwarf mutant, chiquita1-1 (chiq1-1, also called constitutively stressed 1 (cost1)), by growing mutant seedlings together with the wild-type to ensure uniform soil water availability across genotypes. Our results demonstrate that the dwarf phenotype conferred by loss of CHIQ1 function results in constitutively lower water usage per plant, but not increased drought resistance. Our study provides an easily reproducible, low-cost method to measure and control for SWC and to compare drought-resistant genotypes more accurately.

59 BASIC BIOLOGICAL SCIENCES↗