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. Non-crew time 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), integration readiness level (IRL), 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.

Paternal imprinting of dosage-effect defective1 contributes to seed weight xenia in maize.

Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinted dosage-effect defective1 (ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5-10% seed weight reduction when ded1 is transmitted through the male, while homozygous mutants are defective with a 70-90% seed weight reduction. Ded1 encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting of Ded1 causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

Accelerating Biological Insight for Understudied Genes.

The rapid expansion of genome sequence data is increasing the discovery of protein-coding genes across all domains of life. Annotating these genes with reliable functional information is necessary to understand evolution, to define the full biochemical space accessed by nature, and to identify target genes for biotechnology improvements. The majority of proteins are annotated based on sequence conservation with no specific biological, biochemical, genetic, or cellular function identified. Recent technical advances throughout the biological sciences enable experimental research on these understudied protein-coding genes in a broader collection of species. However, scientists have incentives and biases to continue focusing on well documented genes within their preferred model organism. This perspective suggests a research model that seeks to break historic silos of research bias by enabling interdisciplinary teams to accelerate biological functional annotation. We propose an initiative to develop coordinated projects of collaborating evolutionary biologists, cell biologists, geneticists, and biochemists that will focus on subsets of target genes in multiple model organisms. Concurrent analysis in multiple organisms takes advantage of evolutionary divergence and selection, which causes individual species to be better suited as experimental models for specific genes. Most importantly, multisystem approaches would encourage transdisciplinary critical thinking and hypothesis testing that is inherently slow in current biological research.

Characterization of pea seed nutritional value within a diverse population of Pisum sativum.

Micronutrient malnutrition is a global concern that affects more than two billion people worldwide. Pea (Pisum sativum) is a nutritious pulse crop with potential to assist in tackling hidden hunger. Here we report seed ionomic data of 96 diverse pea accessions collected via inductively coupled plasma mass spectrometry (ICP-MS). We found a 100 g serving of peas provides the following average percent daily value for U.S. recommendations: 8% Ca, 39% Mg, 73% Cu, 37% Fe, 63% Mn, 45% Zn, 28% K, and 43% P. Correlations were observed between the majority of minerals tested suggesting strong interrelationships between mineral concentration levels. Hierarchical clustering identified fifteen accessions with high-ranking mineral concentrations. Thirty accessions could be compared to earlier inductively coupled optical emission spectrometry (ICP-OES) data, which revealed significant differences particularly for elements at extreme low or high levels of accumulation. These results improve our understanding of the range of variation in mineral content found in peas and provide additional mineral data resources for germplasm selection.

Genome assembly and population genomic analysis provide insights into the evolution of modern sweet corn.

Sweet corn is one of the most important vegetables in the United States and Canada. Here, we present a de novo assembly of a sweet corn inbred line Ia453 with the mutated shrunken2-reference allele (Ia453-sh2). This mutation accumulates more sugar and is present in most commercial hybrids developed for the processing and fresh markets. The ten pseudochromosomes cover 92% of the total assembly and 99% of the estimated genome size, with a scaffold N50 of 222.2 Mb. This reference genome completely assembles the large structural variation that created the mutant sh2-R allele. Furthermore, comparative genomics analysis with six field corn genomes highlights differences in single-nucleotide polymorphisms, structural variations, and transposon composition. Phylogenetic analysis of 5,381 diverse maize and teosinte accessions reveals genetic relationships between sweet corn and other types of maize. Our results show evidence for a common origin in northern Mexico for modern sweet corn in the U.S. Finally, population genomic analysis identifies regions of the genome under selection and candidate genes associated with sweet corn traits, such as early flowering, endosperm composition, plant and tassel architecture, and kernel row number. Our study provides a high-quality reference-genome sequence to facilitate comparative genomics, functional studies, and genomic-assisted breeding for sweet corn.

Engineering 6-phosphogluconate dehydrogenase improves grain yield in heat-stressed maize.

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 the peptide coding sequence to the Pgd1 and Pgd2 open reading frames (ORFs). 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 into the 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.

Competitive Growth Assay of Mutagenized Chlamydomonas reinhardtii 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 (ISS) to grow terrestrial plants. Live cultures can be stored for at least 1 month in the bags at room temperature. The gene set required for growth in these photobioreactors was tested using a competitive growth assay with mutations induced by short-wave ultraviolet light (UVC) mutagenesis in either wild-type (CC-5082) or cw15 mutant (CC-1883) strains at the start of the assay. Genome sequencing identified UVC-induced mutations, which were enriched for transversions and non-synonymous 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 that modification of DNA repair, signal transduction, and metabolite transport may be needed to improve growth rates in this spaceflight production system.

Protein, weight, and oil prediction by single-seed near-infrared spectroscopy for selection of seed quality and yield traits in pea (Pisum sativum).

BACKGROUND: Pea (Pisum sativum) is a prevalent cool-season crop that produces seeds valued for their high protein content. Modern cultivars have incorporated several traits that improved harvested yield. However, progress toward improving seed quality has received less emphasis, in part due to the lack of tools for easily and rapidly measuring seed traits. In this study we evaluated the accuracy of single-seed near-infrared spectroscopy (NIRS) for measuring pea-seed weight, protein, and oil content. A total of 96 diverse pea accessions were analyzed using both single-seed NIRS and wet chemistry methods. To demonstrate field relevance, the single-seed NIRS protein prediction model was used to determine the impact of seed treatments and foliar fungicides on the protein content of harvested dry peas in a field trial. RESULTS: External validation of partial least squares (PLS) regression models showed high prediction accuracy for protein and weight (R2 = 0.94 for both) and less accuracy for oil (R2 = 0.74). Single-seed weight was weakly correlated with protein and oil content in contrast with previous reports. In the field study, the single-seed NIRS predicted protein values were within 10 mg g-1 of an independent analytical reference measurement and were sufficiently precise to detect small treatment effects. CONCLUSION: The high accuracy of protein and weight estimation show that single-seed NIRS could be used in the dual selection of high-protein, high-weight peas early in the breeding cycle, allowing for faster genetic advancement toward improved pea nutritional quality. © 2020 Society of Chemical Industry.

EMS Mutagenesis of Maize Pollen.

Effective mutagenesis is critical for connecting traits of interest to specific plant genes. The development of site-directed mutagenesis and sequenced-indexed genetics resources in maize allows for targeted analysis of individual genes. These reverse genetics approaches have the potential for confirmation bias by only studying candidate genes for association with traits of interest. Genetic screens of induced, random mutations are important for identifying novel loci as well as interacting factors for known mutant loci. Chemical mutagenesis provides very high mutation rates and can be used for a variety of screen designs. This chapter provides an updated protocol for ethyl methanesulfonate (EMS) mutagenesis of maize pollen using paraffin or mineral oil. Mutagenesis occurs in mature pollen causing nonconcordant endosperm and embryo genotypes as well as sectored M1 plants. Considerations for these factors in genetic screens are discussed.

Genetic Screens to Target Embryo and Endosperm Pathways in Arabidopsis and Maize.

The major tissue types and stem-cell niches of plants are established during embryogenesis, and thus knowledge of embryo development is essential for a full understanding of plant development. Studies of seed development are also important for human health, because the nutrients stored in both the embryo and endosperm of plant seeds provide an essential part of our diet. Arabidopsis and maize have evolved different types of seeds, opening a range of experimental opportunities. Development of the Arabidopsis embryo follows an almost invariant pattern, while cell division patterns of maize embryos are variable. Embryo-endosperm interactions are also different between the two species: in Arabidopsis, the endosperm is consumed during seed development, while mature maize seeds contain an enormous endosperm. Genetic screens have provided important insights into seed development in both species. In the genomic era, genetic analysis will continue to provide important tools for understanding embryo and endosperm biology in plants, because single gene functional studies can now be integrated with genome-wide information. Here, we lay out important factors to consider when designing genetic screens to identify new genes or to probe known pathways in seed development. We then highlight the technical details of two previous genetic screens that may serve as useful examples for future experiments.

Maize defective kernel5 is a bacterial TamB homologue required for chloroplast envelope biogenesis.

Chloroplasts are of prokaryotic origin with a double-membrane envelope separating plastid metabolism from the cytosol. Envelope membrane proteins integrate chloroplasts with the cell, but envelope biogenesis mechanisms remain elusive. We show that maize defective kernel5 (dek5) is critical for envelope biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. The DEK5 protein is homologous to rice SSG4, Arabidopsis thaliana EMB2410/TIC236, and Escherichia coli tamB. TamB functions in bacterial outer membrane biogenesis. DEK5 is localized to the 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. Proteomics and antibody-based analyses show dek5 reduces levels of Toc75 and 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 transport of metabolites and proteins.

Effects of long-term exposure to elevated temperature on Zea mays endosperm development during grain fill.

Cereal yields decrease when grain fill proceeds under conditions of prolonged, moderately elevated temperatures. Endosperm-endogenous processes alter both rate and duration of dry weight gain, but underlying mechanisms remain unclear. Heat effects could be mediated by either abnormal, premature cessation of storage compound deposition or accelerated implementation of normal development. This study used controlled environments to isolate temperature as the sole environmental variable during Zea mays kernel-fill, from 12 days after pollination to maturity. Plants subjected to elevated day, elevated night temperatures (38°C day, 28°C night (38/28°C])) or elevated day, normal night (38/17°C), were compared with those from controls grown under normal day and night conditions (28/17°C). Progression of change over time in endosperm tissue was followed to dissect contributions at multiple levels, including transcriptome, metabolome, enzyme activities, product accumulation, and tissue ultrastructure. Integrated analyses indicated that the normal developmental program of endosperm is fully executed under prolonged high-temperature conditions, but at a faster rate. Accelerated development was observed when both day and night temperatures were elevated, but not when daytime temperature alone was increased. Although transcripts for most components of glycolysis and respiration were either upregulated or minimally affected, elevated temperatures decreased abundance of mRNAs related to biosynthesis of starch and storage proteins. Further analysis of 20 central-metabolic enzymes revealed six activities that were reduced under high-temperature conditions, indicating candidate roles in the observed reduction of grain dry weight. Nonetheless, a striking overall resilience of grain filling in the face of elevated temperatures can be attributed to acceleration of normal endosperm development.

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 (Zea mays) RNA binding motif protein 48 (RBM48) is a U12 splicing factor that functions to promote cell differentiation and repress cell proliferation. RBM48 is coselected with the U12 splicing factor, zinc finger CCCH-type, RNA binding motif, and Ser/Arg rich 2/Rough endosperm 3 (RGH3). Protein-protein interactions between RBM48, RGH3, and U2 Auxiliary Factor (U2AF) subunits suggest major and minor spliceosome factors required for intron recognition form complexes with RBM48. Human RBM48 interacts with armadillo repeat containing 7 (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.

Modulation of early maize seedling performance via priming under sub-optimal temperatures.

Seeds planted in early spring frequently experience low temperature stress in the soil during germination and early plant growth. Seed pretreatments such as priming have been shown to ameliorate the negative effects of cold soil in some crops. However, the potential beneficial effects of priming have not been widely investigated for Zea mays (maize). To investigate seed priming effects, 24 diverse maize inbred lines were primed using a synthetic solid matrix, Micro-Cel E, and then exposed to 10°C soil conditions. Six DSLR cameras captured time lapsed images of emerging seedlings. Manual scoring was used to determine treatment effects on three seedling emergence metrics. Chilling substantially reduced total emergence for two of 24 genotypes evaluated. For these genotypes, priming provided protection allowing nearly full emergence. Priming significantly reduced mean emergence time and increased the emergence uniformity of chilling sensitive genotypes. The results suggest that the cold sensitive genotypes may benefit from priming pretreatment. Kernel density, weight, oil, protein, and starch traits, as determined by single-kernel near infrared spectroscopy, were not correlated with seedling emergence traits supporting a conclusion that early seedling performance cannot be determined from these maize kernel characteristics.

The maize W22 genome provides a foundation for functional genomics and transposon biology.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

Ovary abortion is prevalent in diverse maize inbred lines and is under genetic control.

Crop improvement programs focus on characteristics that are important for plant productivity. Typically genes underlying these traits are identified and stacked to create improved cultivars. Hence, identification of valuable traits for plant productivity is critical for plant improvement. Here we describe an important characteristic for maize productivity. Despite the fact mature maize ears are typically covered with kernels, we find that only a fraction of ovaries give rise to mature kernels. Non-developed ovaries degenerate while neighboring fertilized ovaries produce kernels that fill the ear. Abortion occurs throughout the ear, not just at the tip. We show that the fraction of aborted ovaries/kernels is genetically controlled and varies widely among maize lines, and low abortion genotypes are rare. Reducing or eliminating ovary abortion could substantially increase yield, making this characteristic a new target for selection in maize improvement programs.

Quantitative trait loci associated with soybean seed weight and composition under different phosphorus levels.

Seed size and composition are important traits in food crops and can be affected by nutrient availability in the soil. Phosphorus (P) is a non-renewable, essential macronutrient, and P deficiency limits soybean (Glycine max) yield and quality. To investigate the associations of seed traits in low- and high-P environments, soybean recombinant inbred lines (RILs) from a cross of cultivars Fiskeby III and Mandarin (Ottawa) were grown under contrasting P availability environments. Traits including individual seed weight, seed number, and intact mature pod weight were significantly affected by soil P levels and showed transgressive segregation among the RILs. Surprisingly, P treatments did not affect seed composition or weight, suggesting that soybean maintains sufficient P in seeds even in low-P soil. Quantitative trait loci (QTLs) were detected for seed weight, intact pods, seed volume, and seed protein, with five significant QTLs identified in low-P environments and one significant QTL found in the optimal-P environment. Broad-sense heritability estimates were 0.78 (individual seed weight), 0.90 (seed protein), 0.34 (seed oil), and 0.98 (seed number). The QTLs identified under low P point to genetic regions that may be useful to improve soybean performance under limiting P conditions.

Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize.

Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S) of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer) mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds), Enhancer/Suppressor-mutator (En/Spm), and Mutator (Mu) transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm.

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.

Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development.

RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3 Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates.