Summary: 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.

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.

The starch metabolic network was investigated in relation to other metabolic processes by examining a mutant with altered single-gene expression of ATP citrate lyase (ACL), an enzyme responsible for generating cytosolic acetyl-CoA pool from citrate. Previous research has shown that transgenic antisense plants with reduced ACL activity accumulate abnormally enlarged starch granules. In this study, we explored the underlying molecular mechanisms linking cytosolic acetyl-CoA generation and starch metabolism under short-day photoperiods. We performed transcriptome and quantification of starch accumulation in the leaves of wild-type and antisense seedlings with reduced ACL activity. The antisense-ACLA mutant accumulated more starch than the wild type under short-day conditions. Zymogram analyses were conducted to compare the activities of starch-metabolizing enzymes with transcriptomic changes in the seedling. Differential expression between wild-type and antisense-ACLA plants was detected in genes implicated in starch and acetyl-CoA metabolism, and cell wall metabolism. These analyses revealed a strong correlation between the transcript levels of genes responsible for starch synthesis and degradation, reflecting coordinated regulation at the transcriptomic level. Furthermore, our data provide novel insights into the regulatory links between cytosolic acetyl-CoA metabolism and starch metabolic pathways.

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.