CRISPR/Cas9-based editing of NF-YC4 promoters yields high-protein rice and soybean.

Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts. Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs. Building upon our prior research demonstrating the protein-boosting effects of the transcription factor NF-YC4, we identified conserved motifs targeted by RAV and WRKY repressors in the NF-YC4 promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increased NF-YC4 expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein. Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality.

Orphan Genes in Crop Improvement: Enhancing Potato Tuber Protein without Impacting Yield.

Qua-Quine Starch (QQS), an Arabidopsis thaliana orphan gene, and its interactor, Arabidopsis Nuclear Factor Y subunit C4 (AtNF-YC4), can increase the total leaf and seed protein in different plants. Despite their potential in developing protein-rich crop varieties, their influence on the protein content of the stem, modified stem, and tuber was never investigated. Potato (Solanum tuberosum) is one of the most valuable food crops worldwide. This staple food is rich in starch, vitamins (B6, C), phenolics, flavonoids, polyamines, carotenoids, and various minerals but lacks adequate proteins necessary for a healthy human diet. Here we expressed A. thaliana QQS (AtQQS) and overexpressed S. tuberosum NF-YC4 (StNF-YC4) in potatoes to determine their influence on the composition and morphological characteristics of potato tubers. Our data demonstrated higher protein and reduced starch content in potato tubers without significantly compromising the tuber yield, shape, and numbers, when QQS was expressed or StNF-YC4 was overexpressed. Publicly available expression data, promoter region, and protein−protein interaction analyses of StNF-YC4 suggest its potential functionality in potato storage protein, metabolism, stress resistance, and defense against pests and pathogens. The overall outcomes of this study support QQS and NF-YC4's potential utilization as tools to enhance tuber protein content in plants.

AtQQS orphan gene and NtNF-YC4 boost protein accumulation and pest resistance in tobacco (Nicotiana tabacum).

Qua-Quine Starch (QQS), an orphan gene exclusively found in Arabidopsis thaliana, interacts with Nuclear Factor Y subunit C4 (NF-YC4) and regulates carbon and nitrogen allocation in different plant species. Several studies uncovered its potential in increasing total protein and resistance against pathogens/pests in Arabidopsis and soybean. However, it is still unclear if these attributes QQS offers are universal in all flowering plants. Here we studied AtQQS and Nicotiana tabacum NF-YC4's (NtNF-YC4) influence on starch/protein content and pest resistance in tobacco. Our results showed both AtQQS and NtNF-YC4 had a positive impact on the plant's total protein accumulation. Simultaneously, we have also observed reduced starch biosynthesis and increased resistance against common pests like whiteflies (Bemisia tabaci) and aphids (Myzus persicae) in tobacco plants expressing AtQQS or overexpressing NtNF-YC4. Real-time PCR also revealed increased NF-YC4 expression after aphid infestation in tobacco varieties with higher pest resistance but decreased/unchanged NF-YC4 expression in varieties susceptible to pests. Further analysis revealed that QQS expression and overexpression of NtNF-YC4 strongly repressed expression of genes such as sugar transporter SWEET10 and Flowering Locus T (FT), suggesting involvement of SWEET10 and FT in the QQS and NF-YC4 mediated carbon and nitrogen allocation in tobacco. Our data suggested that the activity of species-specific orphan genes may not be limited to the original species or its close relatives. Sequence alignment revealed the conserved sequence of the NF-YC4s in different plant species that may be responsible for the resulting shift in metabolism, pest resistance. Cis-acting DNA element analysis of NtNF-YC4 promoter region may outline potential mechanisms for these phenotypic changes.

GmNF-YC4-2 Increases Protein, Exhibits Broad Disease Resistance and Expedites Maturity in Soybean.

The NF-Y gene family is a highly conserved set of transcription factors. The functional transcription factor complex is made up of a trimer between NF-YA, NF-YB, and NF-YC proteins. While mammals typically have one gene for each subunit, plants often have multigene families for each subunit which contributes to a wide variety of combinations and functions. Soybean plants with an overexpression of a particular NF-YC isoform GmNF-YC4-2 (Glyma.04g196200) in soybean cultivar Williams 82, had a lower amount of starch in its leaves, a higher amount of protein in its seeds, and increased broad disease resistance for bacterial, viral, and fungal infections in the field, similar to the effects of overexpression of its isoform GmNF-YC4-1 (Glyma.06g169600). Interestingly, GmNF-YC4-2-OE (overexpression) plants also filled pods and senesced earlier, a novel trait not found in GmNF-YC4-1-OE plants. No yield difference was observed in GmNF-YC4-2-OE compared with the wild-type control. Sequence alignment of GmNF-YC4-2, GmNF-YC4-1 and AtNF-YC1 indicated that faster maturation may be a result of minor sequence differences in the terminal ends of the protein compared to the closely related isoforms.

QQS orphan gene and its interactor NF-YC4 reduce susceptibility to pathogens and pests.

Enhancing the nutritional quality and disease resistance of crops without sacrificing productivity is a key issue for developing varieties that are valuable to farmers and for simultaneously improving food security and sustainability. Expression of the Arabidopsis thaliana species-specific AtQQS (Qua-Quine Starch) orphan gene or its interactor, NF-YC4 (Nuclear Factor Y, subunit C4), has been shown to increase levels of leaf/seed protein without affecting the growth and yield of agronomic species. Here, we demonstrate that overexpression of AtQQS and NF-YC4 in Arabidopsis and soybean enhances resistance/reduces susceptibility to viruses, bacteria, fungi, aphids and soybean cyst nematodes. A series of Arabidopsis mutants in starch metabolism were used to explore the relationships between QQS expression, carbon and nitrogen partitioning, and defense. The enhanced basal defenses mediated by QQS were independent of changes in protein/carbohydrate composition of the plants. We demonstrate that either AtQQS or NF-YC4 overexpression in Arabidopsis and in soybean reduces susceptibility of these plants to pathogens/pests. Transgenic soybean lines overexpressing NF-YC4 produce seeds with increased protein while maintaining healthy growth. Pull-down studies reveal that QQS interacts with human NF-YC, as well as with Arabidopsis NF-YC4, and indicate two QQS binding sites near the NF-YC-histone-binding domain. A new model for QQS interaction with NF-YC is speculated. Our findings illustrate the potential of QQS and NF-YC4 to increase protein and improve defensive traits in crops, overcoming the normal growth-defense trade-offs.

QQS orphan gene regulates carbon and nitrogen partitioning across species via NF-YC interactions.

The allocation of carbon and nitrogen resources to the synthesis of plant proteins, carbohydrates, and lipids is complex and under the control of many genes; much remains to be understood about this process. QQS (Qua-Quine Starch; At3g30720), an orphan gene unique to Arabidopsis thaliana, regulates metabolic processes affecting carbon and nitrogen partitioning among proteins and carbohydrates, modulating leaf and seed composition in Arabidopsis and soybean. Here the universality of QQS function in modulating carbon and nitrogen allocation is exemplified by a series of transgenic experiments. We show that ectopic expression of QQS increases soybean protein independent of the genetic background and original protein content of the cultivar. Furthermore, transgenic QQS expression increases the protein content of maize, a C4 species (a species that uses 4-carbon photosynthesis), and rice, a protein-poor agronomic crop, both highly divergent from Arabidopsis. We determine that QQS protein binds to the transcriptional regulator AtNF-YC4 (Arabidopsis nuclear factor Y, subunit C4). Overexpression of AtNF-YC4 in Arabidopsis mimics the QQS-overexpression phenotype, increasing protein and decreasing starch levels. NF-YC, a component of the NF-Y complex, is conserved across eukaryotes. The NF-YC4 homologs of soybean, rice, and maize also bind to QQS, which provides an explanation of how QQS can act in species where it does not occur endogenously. These findings are, to our knowledge, the first insight into the mechanism of action of QQS in modulating carbon and nitrogen allocation across species. They have major implications for the emergence and function of orphan genes, and identify a nontransgenic strategy for modulating protein levels in crop species, a trait of great agronomic significance.