Meta-analyses and theory show that with rising atmospheric [CO2], Rubisco has become the greatest limitation to light-saturated leaf CO2 assimilation rates (Asat) in C4 crops. So would transgenically increasing Rubisco increase Asat and result in increased productivity in the field? Here, we successfully overexpressed the Rubisco small subunit (RbcS) with Rubisco accumulation factor 1 (Raf1) in both sorghum and sugarcane, resulting in significant increases in Rubisco content of 13 to 25% and up to 90% respectively. Asat increased 12 to 15% and Rubisco enzyme activity ~40% in three independent transgenic events of both species. Sorghum plants also showed increased speeds of photosynthetic induction and decreased bundle sheath leakiness. These improvements translated into average increases of 15.5% in biomass in field-grown sorghum and a 37 to 81% increase in greenhouse-grown sugarcane. This suggests a potential opportunity to achieve substantial increases in productivity of this key economically important clade of C4 crops, future proofing their value under global atmospheric change.

In‐context promoter bashing via genome editing is a route to identify and characterize critical regulatory regions that govern expression of genes of interest. The outcomes of in‐context promoter bashing can be used to inform editing strategies to modulate the expression of selected gene models in a desired fashion. Here, we employed in‐context promoter bashing to characterize the proximal upstream regulatory regions of sorghum genes encoding phosphoenolpyruvate carboxykinase bundle sheath ( Sb PEPCK.BS, SbiTx430.01G455400) and alanine aminotransferase bundle sheath ( Sb AlaAT.BS, SbiTx430.02G006600), two proteins involved in the PCK C 4 pathway. Characterized germinal edits within the targeted regions upstream of these two genes ranged in size from 138 up to 1790 bp. A 138 bp within the Sb PEPCK.BS upstream region and a 1643 bp element within the Sb AlaAT.BS upstream region were determined to be important for maintenance of transcription levels. No change in development or various physiological parameters was observed in characterized lineages carrying promoter edits. However, significant changes in seed reserves and a reduction in 100‐seed weight were consistently observed, under both greenhouse and field environments, in plants carrying an edit in the promoter of Sb PEPCK.BS gene, which were significantly reduced in transcript accumulation for this gene.

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Engineering lower stomatal density to reduce stomatal conductance (g(s)) has improved WUE in multiple C-3 crop species. However, reducing g(s) in C-3 species often reduces photosynthetic carbon gain. A different response is expected in C-4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the relationship of photosynthesis (A(N)) with intracellular CO2 concentration (c(i)), such that photosynthesis is CO2 saturated and reductions in g(s) are unlikely to limit A(N). To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C-4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum led to reduced stomatal densities, reduced g(s), reduced plant water use, and avoidance of stress during a period of water deprivation. In addition, moderate reduction in stomatal density did not increase stomatal limitation to A(N). However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues could provide a pathway to improved agronomic outcomes.

Abstract This strategic plan summarizes the major accomplishments achieved in the last quinquennial by the soybean [Glycine max (L.) Merr.] genetics and genomics research community and outlines key priorities for the next 5 years (2024–2028). This work is the result of deliberations among over 50 soybean researchers during a 2‐day workshop in St Louis, MO, USA, at the end of 2022. The plan is divided into seven traditional areas/disciplines: Breeding, Biotic Interactions, Physiology and Abiotic Stress, Functional Genomics, Biotechnology, Genomic Resources and Datasets, and Computational Resources. One additional section was added, Training the Next Generation of Soybean Researchers, when it was identified as a pressing issue during the workshop. This installment of the soybean genomics strategic plan provides a snapshot of recent progress while looking at future goals that will improve resources and enable innovation among the community of basic and applied soybean researchers. We hope that this work will inform our community and increase support for soybean research.

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (gsw). However, the quantitative relationship between reduced SD, gsw, and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEPF2) in sugarcane reduced SD by 26-38% but did not affect gsw compared with the wild type. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C3 dicots, C3 monocots, and C4 monocots revealed that engineered reductions in SD are strongly correlated with lower gsw (r2=0.60-0.98), but this response is damped relative to the change in anatomy.

A reliable and simple Agrobacterium -mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II ( nptII ) and the phleomycin resistance ( bleI ) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens . The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 10 6 cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes.

Microtubule-associated protein 65-1 (MAP65-1) protein plays an essential role in plant cellular dynamics through impacting stabilization of the cytoskeleton by serving as a crosslinker of microtubules. The role of MAP65-1 in plants has been associated with phenotypic outcomes in response to various environmental stresses. The Arabidopsis MAP65-1 (AtMAP65-1) is a known virulence target of plant bacterial pathogens and is thus a component of plant immunity. Soybean events were generated that carry transgenic alleles for both AtMAP65-1 and GmMAP65-1, the soybean AtMAP65-1 homolog, under control of cauliflower mosaic virus 35S promoter. Both AtMAP65-1 and GmMAP65-1 transgenic soybeans are more resistant to challenges by the soybean bacterial pathogen Pseudomonas syringae pv. glycinea and the oomycete pathogen Phytophthora sojae, but not the soybean cyst nematode, Heterodera glycines. Soybean plants expressing AtMAP65-1 and GmMAP65-1 also display a tolerance to the herbicide oryzalin, which has a mode of action to destabilize microtubules. In addition, GmMAP65-1-expressing soybean plants show reduced cytosol ion leakage under freezing conditions, hinting that ectopic expression of GmMAP65-1 may enhance cold tolerance in soybean. Taken together, overexpression of AtMAP65-1 and GmMAP65-1 confers tolerance of soybean plants to various biotic and abiotic stresses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Maize grain is deficient in lysine. While the opaque2 mutation increases grain lysine, o2 is a transcription factor that regulates a wide network of genes beyond zeins, which leads to pleiotropic and often negative effects. Additionally, the drastic reduction in 19 kDa and 22 kDa alpha-zeins causes a floury kernel, unsuitable for agricultural use. Quality protein maize (QPM) overcame the undesirable kernel texture through the introgression of modifying alleles. However, QPM still lacks a functional o2 transcription factor, which has a penalty on non-lysine amino acids due to the o2 mutation. CRISPR/cas9 gives researchers the ability to directly target genes of interest. In this paper, gene editing was used to specifically target the 19 kDa alpha zein gene family. This allows for proteome rebalancing to occur without an o2 mutation and without a total alpha-zein knockout. The results showed that editing some, but not all, of the 19 kDa zeins resulted in up to 30% more lysine. An edited line displayed an increase of 30% over the wild type. While not quite the 55% lysine increase displayed by QPM, the line had little collateral impact on other amino acid levels compared to QPM. Additionally, the edited line containing a partially reduced 19 kDa showed an advantage in kernel texture that had a complete 19 kDa knockout. These results serve as proof of concept that editing the 19 kDa alpha-zein family alone can enhance lysine while retaining vitreous endosperm and a functional O2 transcription factor.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans.Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment.Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil).Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans.

Cas9-based genome editing is a powerful genetic tool for loci specifically targeted for genome modification. This chapter describes up-to-date protocols using Cas9-based genome editing technology, including vector construction with GoldenBraid assembly, Agrobacterium-mediated soybean transformation, and identification of editing in the genome.

Summary Soybean oil is one of the most consumed vegetable oils worldwide. Genetic improvement of its concentration in seeds has been historically pursued due to its direct association with its market value. Engineering attempts aiming to increase soybean seed oil presented different degrees of success that varied with the genetic design and the specific variety considered. Understanding the embryo’s responses to the genetic modifications introduced, is a critical step to successful approaches. In this work, the metabolic and transcriptional responses to AtWRI1 and AtDGAT1 expression in soybean seeds were evaluated. AtWRI1 is a master regulator of fatty acid (FA) biosynthesis, and AtDGAT1 encodes an enzyme catalysing the final and rate‐limiting step of triacylglycerides biosynthesis. The events expressing these genes in the embryo did not show an increase in total FA content, but they responded with changes in the oil and carbohydrate composition. Transcriptomic studies revealed a down‐regulation of genes putatively encoding for oil body packaging proteins, and a strong induction of genes annotated as lipases and FA biosynthesis inhibitors. Novel putative AtWRI1 targets, presenting an AW‐box in the upstream region of the genes, were identified by comparison with an event that harbours only AtWRI1. Lastly, targeted metabolomics analysis showed that carbon from sugar phosphates could be used for FA competing pathways, such as starch and cell wall polysaccharides, contributing to the restriction in oil accumulation. These results allowed the identification of key cellular processes that need to be considered to break the embryo’s natural restriction to uncontrolled seed lipid increase.

There are three components that impact crop harvests: the genetics of the seed sown, the environment in which the crop is grown, and the inputs employed during the growing period. For most species, the genetic component is addressed through breeding programs. Breeding programs require genetic variation within the available crop population from which parental crosses are made. Breeding programs that develop elite cultivars for commodities such as maize, soybean, wheat and sorghum, have wonderful inherent genetic diversity to utilise. However, many desired traits that breeders seek are not present within existing crop populations. It is here where the tools of biotechnology can complement plant breeding programs, by introducing novel pieces of genetic variation that can impart these favourable traits.

SUMMARYSoybean [Glycine max (L.) Merr.] is a unique crop species because it has high levels of both protein and oil in its seed. Of the many quantitative trait loci (QTL) controlling soybean seed protein content, alleles of the cqSeed protein‐003 QTL on chromosome 20 exert the greatest additive effect. The high‐protein allele exists in both cultivated and wild soybean (Glycine soja Siebold & Zucc.) germplasm. Our objective was to fine map this QTL to enable positional‐based cloning of its underlying causative gene(s). Fine mapping was achieved by developing and testing a series of populations in which the chromosomal region surrounding the segregating high‐ versus low‐protein alleles was gradually narrowed, using marker‐based detection of recombinant events. The resultant 77.8 kb interval was directly sequenced from a G. soja source and compared with the reference genome to identify structural and sequence polymorphisms. An insertion/deletion variant detected in Glyma.20G85100 was found to have near‐perfect +/− concordance with high/low‐protein allele genotypes inferred for this QTL in parents of published mapping populations. The indel structure was concordant with an evolutionarily recent insertion of a TIR transposon into the gene in the low‐protein lineage. Seed protein was significantly greater in soybean expressing an RNAi hairpin downregulation element in two independent events relative to control null segregant lineages. We conclude that a transposon insertion within the CCT domain protein encoded by the Glyma.20G85100 gene accounts for the high/low seed protein alleles of the cqSeed protein‐003 QTL.

In this study, the economics of producing biofuels from an industrial hemp (Cannabis sativa) genotype e 19m96136 was investigated. A lignocellulosic biofuel plant, hourly consuming 85 metric tons of hemp biomass was modeled in SuperPro Designer (R). The integrated bioenergy plant produced hemp biodiesel and bioethanol from lipids and carbohydrates, respectively. The structural composition of the industrial hemp plant was analyzed in a previous study. The data obtained was used to simulate feedstock composition in SuperPro Designer (R). The simulation results indicated that Hemp containing 2% lipids can yield up to 3.95 million gallons of biodiesel annually. On improving biomass lipid content to 5 and 10%, biodiesel production increased to 9.88 and 19.91 million gallons, respectively. The breakeven unit production cost of hemp biodiesel with 2, 5, and 10% lipid containing hemp was $18.49, $7.87, and $4.13/gallon, respectively. The biodiesel unit production cost when utilizing 10% lipid-containing hemp was comparable to soybean biodiesel at $4.13/gallon. Furthermore, sensitivity analysis revealed the possibility of a 7.80% reduction in unit production cost upon a 10% reduction in hemp feedstock cost. Furthermore, industrial hemp was capable of producing between 307.80 and 325.82 gallons of total biofuels per hectare of agricultural land than soybean. (C) 2021 Elsevier Ltd. All rights reserved.

AbstractBackgroundBiological mutagens (such as transposon) with sequences inserted, play a crucial role to link observed phenotype and genotype in reverse genetic studies. For this reason, accurate and efficient software tools for identifying insertion sites based on the analysis of sequencing reads are desired.ResultsWe developed a bioinformatics tool, a Finder, to identify genome-wide Insertions in Mutagenesis (named as “InMut-Finder”), based on target sequences and flanking sequences from long reads, such as Oxford Nanopore Sequencing. InMut-Finder succeeded in identify > 100 insertion sites inMedicago truncatulaand soybean mutants based on sequencing reads of whole-genome DNA or enriched insertion-site DNA fragments. Insertion sites discovered by InMut-Finder were validated by PCR experiments.ConclusionInMut-Finder is a comprehensive and powerful tool for automated insertion detection from Nanopore long reads. The simplicity, efficiency, and flexibility of InMut-Finder make it a valuable tool for functional genomics and forward and reverse genetics. InMut-Finder was implemented with Perl, R, and Shell scripts, which are independent of the OS. The source code and instructions can be accessed athttps://github.com/jsg200830/InMut-Finder.

Additional file 2: Supplemental Table S1. All insertion sites identified in one soybean Ds mutant. Supplemental Table S2. Three selected insertion sites and primer sequences for PCR validation for the soybean mutant line. Supplemental Table S3. All insertion sites identified in one Tnt1 mutant of Medicago truncatula.