Biology subjects

Settles, A. M.

Publications and source records attributed to Settles, A. M..

10 recordsLinked to original sources

bioRxiv2026

Single-kernel near-infrared spectroscopy enables haploid kernel sorting in field and sweet corn using high-oil haploid inducers across diverse donor-inducer combinations

Key messageA single-kernel near-infrared reflectance spectroscopy-based sorter can effectively identify haploid kernels for doubled haploid production in field and sweet corn backgrounds. Doubled haploid (DH) technology significantly shortens the breeding cycle for developing homozygous inbred lines in maize (Zea mays). Manual sorting of haploids from a larger bulk of hybrid kernels in an induction cross is a major bottleneck in DH development. Automated systems based on near-infrared (NIR) reflectance spectroscopy can be valuable tools for rapid haploid sorting, provided that sorting accuracy is sufficient for incorporation into the DH process. In this study, we evaluated the accuracy of a custom-built single-kernel NIR (skNIR) sorter for classifying haploid kernels from 12 high-oil haploid induction populations generated from two sweet corn and two field corn donors and four high-oil haploid inducers (HOHIs). We evaluated several general classification models that can be applied without population-specific recalibration or prior genotyping, including models that classified haploids based solely on predicted oil content, as well as multivariate methods that used all wavelengths of the NIR spectra. The highest classification accuracy was obtained using a general multivariate support vector machine (SVM) model. When combined with the two best-performing HOHIs, the general SVM model accurately sorted induction populations from two of the three donor backgrounds crossed with these inducers. Two oil-based methods showed less accurate classification than the multivariate SVM model, due to overlapping oil content distributions across the two kernel classes. Overall, this study demonstrates effective skNIR-based sorting of haploid kernels from diverse induction populations using a single general model. The practical deployment of this instrument in maize breeding programs is discussed.

plant biology↗

bioRxiv2025

Efficient long-term cryopreservation of Arthrospira platensis NIES-39 and adaptation for the Space Algae-2 spaceflight experiment

Arthrospira platensis, commonly known as spirulina, is a photosynthetically efficient cyanobacterium used for production of nutritional supplements, biofuel, wastewater treatment, and CO2 sequestration. These traits and uses make it a candidate for bioregenerative life support systems for space travel and future habitats. However, A. platensis lacks a cryopreservation method for long-term storage of viable cells, and genetic stocks are currently sub-cultured for maintenance. Here we report an efficient cryopreservation method for A. platensis strain NIES-39 and adapt this method for an upcoming spaceflight experiment. Seven cryoprotective agents were tested to preserve viable A. platensis. We found that a combination of dimethyl sulfoxide and trehalose additives to Zarrouks media protected trichome viability for extended storage periods at -80 {degrees}C. Throughout one year of frozen storage, we observed equivalent viability of cryopreserved cells in comparison to non-frozen cultures. Equivalent cryopreservation was demonstrated with multiple volumes of frozen culture, culture containers, and thawing methods to adapt the method for delivery and return of viable NIES-39 for the Space Algae-2 experiment on the International Space Station. The method requires minimal resources, employs simple freezing and thawing procedures, and could be implemented in commercial production to preserve genetic stocks.

microbiology↗

bioRxiv2024

Maize rough endosperm6 is a predicted RNA helicase required for miRNA processing and endosperm cell patterning

Maize rough endosperm mutants have defective kernels with a rough, etched, or pitted endosperm surface. Molecular genetic analysis of this mutant class has identified multiple RNA processing proteins critical to endosperm development. Here, we report that rough endosperm6 (rgh6) encodes a predicted DEAD-box RNA helicase required for miRNA processing. Mutant rgh6 kernels reduce grain fill and increase relative transcript levels of markers specific to epidermal endosperm cell types. B-A translocation crosses revealed that rgh6 mutant endosperm inhibits normal embryo development. We mapped the rgh6 locus to a three gene interval and confirmed it encodes a predicted DEAD-box RNA helicase with two independent transposon-tagged alleles. Transient expression of a RGH6-green fluorescent protein (GFP) fusion is localized to nucleolus and nuclear speckles consistent with a function in nuclear RNA processing. Mutant rgh6 endosperms have increased precursor miRNA and decreased mature miRNA levels indicating that rgh6 impacts miRNA processing. Our study demonstrates that precursor miRNA processing and miRNA target regulation are required for normal endosperm development.

plant biology↗

bioRxiv2024

Integrating Phenomic Selection Using Single-Kernel Near-Infrared Spectroscopy and Genomic Selection for Corn Breeding Improvement

Phenomic Selection (PS) is a cost-effective method proposed for predicting complex traits and enhancing genetic gain in breeding programs. The statistical procedures are similar to those utilized in genomic selection (GS) models, but molecular markers data are replaced with phenomic data, such as near-infrared spectroscopy (NIRS). However, the use of NIRS applied to PS typically utilized destructive sampling or collected data after the establishment of selection experiments in the field. Here, we explored the application of PS using non-destructive, single-kernel NIRS in a sweet corn breeding program, focusing on predicting future, unobserved field-based traits of economic importance, including ear and vegetative traits. Three models were employed on a diversity panel: G-BLUP and P-BLUP models, which used relationship matrices based on SNP and NIRS data, and a combined model. The genomic relationship matrices were evaluated with varying numbers of SNPs. Additionally, the P-BLUP model trained on the diversity panel was used to select doubled haploid (DH) lines for germination before planting, with predictions validated using observed data. The findings indicate that PS generated good predictive ability (e.g., 0.46 for plant height) and effectively distinguished between high and low germination rates in untested DH lines. Although GS generally outperformed PS, the model combining both information yielded the highest predictive ability, with considerably higher accuracies than GS when low marker densities were used. This study highlights the potential of NIRS both to achieve genetic gain where GS may not be feasible and to maintain/improve accuracy with SNP-based information while reducing genotyping costs. Key messagePhenomic selection using whole seeds is a promising alternative to improve genetic gain and complement genomic selection in corn breeding. Models that combine genomic and phenomic data maximize the predictive ability.

genetics↗

bioRxiv2022

Theoretical design of a space bioprocessing system to produce recombinant proteins

Space-based biomanufacturing has the potential to improve the sustainability of deep space exploration. To advance biomanufacturing, bioprocessing systems need to be developed for space applications. Here, commercial technologies were assessed to design space bioprocessing systems to supply a liquid amine carbon dioxide scrubber with active carbonic anhydrase produced recombinantly. Design workflows encompassed biomass dewatering of 1 L Escherichia coli cultures through to recombinant protein purification. Equivalent system mass (ESM) analyses had limited utility for selecting specific technologies. Instead, bioprocessing system designs focused on minimizing complexity and enabling system versatility. Three designs that differed in biomass dewatering and protein purification approaches had nearly equivalent ESM of 357-522 kg eq. Values from the system complexity metric (SCM), technology readiness level (TRL), and degree of crew assistance metric identified a simpler, less costly, and easier to operate design for automated biomass dewatering, cell lysis, and protein affinity purification.

bioengineering↗

bioRxiv2020

Evolutionary Conservation and Essential Function of Human and Maize RNA Binding Motif Protein 48 (RBM48) in U12-Type Intron Splicing

U12-type or minor introns are found in most multicellular eukaryotes and constitute [~]0.5% of all introns in species with a minor spliceosome. Although the biological significance for evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome (MDS), predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA Binding Motif Protein48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5 end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron containing genes (MIGs) have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of MIGs disrupted in both species. Mutations in the majority of these orthologous MIGs have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.

evolutionary biology↗

bioRxiv2020

Engineering 6-phosphogluconate dehydrogenase to improve heat tolerance in maize seed development

Endosperm starch synthesis is a primary determinant of grain yield and is sensitive to high temperature stress. The maize chloroplast-localized 6-phosphogluconate dehydrogenase (6PGDH), PGD3, is critical for endosperm starch accumulation. Maize also has two cytosolic isozymes, PGD1 and PGD2 that are not required for kernel development. We found that cytosolic PGD1 and PGD2 isozymes have heat stable activity, while amyloplast-localized PGD3 activity is labile under heat stress conditions. We targeted heat-stable 6PGDH to endosperm amyloplasts by fusing the Waxy1 chloroplast targeting peptide coding sequence to the Pgd1 and Pgd2 open reading frames. These WPGD1 and WPGD2 fusion proteins import into isolated chloroplasts demonstrating a functional targeting sequence. Transgenic maize plants expressing WPGD1 and WPGD2 with an endosperm specific promoter increased 6PGDH activity with enhanced heat stability in vitro. WPGD1 and WPGD2 transgenes complement the pgd3 defective kernel phenotype indicating the fusion proteins are targeted to the amyloplast. In the field, the WPGD1 and WPGD2 transgenes can mitigate grain yield losses in high nighttime temperature conditions by increasing kernel number. These results provide insight on subcellular distribution of metabolic activities in the endosperm and suggest the amyloplast pentose phosphate pathway is a heat-sensitive step in maize kernel metabolism that contributes to yield loss during heat stress. Significance StatementHeat stress reduces yield in maize by affecting the number of kernels that develop and the accumulation of seed storage molecules during grain fill. Climate change is expected to increase frequency and duration of high temperature stress, which will lower grain yields. Here we show that one enzyme in central carbon metabolism is sensitive to high temperatures. By providing a heat-resistant form of the enzyme in the correct subcellular compartment, a larger number of kernels develop per plant during heat stress in the field. This genetic improvement could be included as part of integrated approaches to mitigate yield losses due to climate change.

plant biology↗

bioRxiv2020

Selecting for Chlamydomonas reinhardtii fitness in a liquid algal growth system compatible with the International Space Station Veggie plant growth chamber

A biological life support system for spaceflight would capture carbon dioxide waste produced by living and working in space to generate useful organic compounds. Photosynthesis is the primary mechanism to fix carbon into organic molecules. Microalgae are highly efficient at converting light, water, and carbon dioxide into biomass, particularly under limiting, artificial light conditions that are a necessity in space photosynthetic production. Although there is great promise in developing algae for chemical or food production in space, most spaceflight algae growth studies have been conducted on solid agar-media to avoid handling liquids in microgravity. Here we report that breathable plastic tissue culture bags can support robust growth of Chlamydomonas reinhardtii in the Veggie plant growth chamber, which is used on the International Space Station to grow terrestrial plants. Live cultures can be stored for at least one month in the bags at room temperature. The gene set required for growth in these photobioreactors was tested through a short-wave ultraviolet light (UVC) mutagenesis and selection experiment with wild-type (CC-5082) and cw15 mutant (CC-1883) strains. Genome sequencing identified UVC-induced mutations, which were enriched for transversions and nonsynonymous mutations relative to natural variants among laboratory strains. Genes with mutations indicating positive selection were enriched for information processing genes related to DNA repair, RNA processing, translation, cytoskeletal motors, kinases, and ABC transporters. These data suggest modification of signal transduction and metabolite transport may be needed to improve growth rates in this spaceflight production system.

plant biology↗

bioRxiv2018

Maize defective kernel5 is a bacterial tamB homolog required for chloroplast envelope biogenesis

Chloroplasts are of prokaryotic origin with a double membrane envelope that separates plastid metabolism from the cytosol. Envelope membrane proteins integrate the chloroplast with the cell, but the biogenesis of the envelope membrane remains elusive. We show that the maize defective kernel5 (dek5) locus is critical for plastid membrane biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. We show that dek5 encodes a protein homologous to rice SUBSTANDARD STARCH GRAIN4 (SSG4) and E.coli tamB. TamB functions in bacterial outer membrane biogenesis. The DEK5 protein is localized to the chloroplast envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Both proteomics and antibody-based analyses show that dek5 chloroplasts have reduced levels of chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable metabolite transport.

plant biology↗

bioRxiv2018

RNA Binding Motif Protein 48 is required for U12 splicing and maize endosperm differentiation

The last eukaryotic common ancestor had two classes of introns that are still found in most eukaryotic lineages. Common U2-type and rare U12-type introns are spliced by the major and minor spliceosomes, respectively. Relatively few splicing factors have been shown to be specific to the minor spliceosome. We found that the maize RNA Binding Motif Protein48 (RBM48) is a U12 splicing factor that functions to promote cell differentiation and repress cell proliferation. RBM48 is coselected with the U12 splicing factor, ZRSR2/RGH3. Protein-protein interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits suggest major and minor spliceosome factors may form complexes during intron recognition. Human RBM48 interacts with ARMC7. Maize RBM48 and ARMC7 have a conserved protein-protein interaction. These data predict that RBM48 is likely to function in U12 splicing throughout eukaryotes and that U12 splicing promotes endosperm cell differentiation in maize.

plant biology↗