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.

Drought stress poses a substantial challenge to plant growth and agricultural productivity worldwide. Upon water depletion, plants activate an abscisic acid (ABA) signaling pathway, leading to stomatal closure to reduce water loss. The MYB family of transcription factors plays diverse roles in growth, development, stress responses and biosynthesis, yet their involvement in stomatal regulation remains unclear. Here, we demonstrate that ABA significantly upregulates the expression of MYB41, MYB74, and MYB102, with MYB41 serving as a key regulator that induces the expression of both MYB74 and MYB102. Through luciferase assays, chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSA), we reveal that MYB41 engages in positive feedback regulation by binding to its own promoter, thus amplifying its transcription in Arabidopsis (Arabidopsis thaliana). Furthermore, our investigation showed that MYB41 recruits BRAHMA (BRM), the core ATPase subunit of the SWI/SNF complex, to the MYB41 promoter, facilitating the binding of HISTONE DEACETYLASE 6 (HDA6). This recruitment triggers epigenetic modifications, resulting in reduced MYB41 expression characterized by elevated H3K27me3 levels and concurrent decreases in H3ac, H3K27ac, and H3K14ac levels in wild-type plants compared to brm knockout mutant plants. Our genetic and molecular analyses show that ABA mediates autoregulation of the MYB41-BRM module, which intricately modulates stomatal movement in A. thaliana. This discovery sheds light on a drought response mechanism with the potential to greatly enhance agricultural productivity.

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.

Taraxacum kok-saghyz Rodin (TKS) is a recognized alternative source of natural rubber comparable to the rubber tree. The geranylgeranyl pyrophosphate synthase (GGPS) catalyzed the synthesis of geranylgeranyl pyrophosphate (GGPP), which is an important enzyme in the secondary metabolism pathway. In this study, we present the first analysis of the GGPS gene family in TKS, where a total of seven TkGGPS family members were identified. Their core motifs, conserved structural domains, gene structures, and cis-acting elements were described. In addition, two phylogenetic trees were constructed based on the Neighbor-Joining and Maximum-Likelihood methods, and the TkGGPSs were highly conserved and exhibited good collinearity with the other species. Transcriptome data showed that seven TkGGPS gene members were expressed in all the 12 tissues measured, and TkGGPS1, TkGGPS3, and TkGGPS6 were highly expressed in latex, suggesting that they may be associated with natural rubber synthesis. Meanwhile, quantitative real-time PCR (qRT-PCR) showed that the expression levels of the TkGGPS genes were regulated by the ethylene and methyl jasmonate (MeJA) pathways. Subcellular localization results indicated that all the TkGGPS proteins were also located in chloroplasts involved in photosynthesis in plants. This study will provide valuable insights into the selection of candidate genes for molecular breeding and natural rubber biosynthesis in TKS.

Cotton fiber length is an important measurement for application in the textile industry, and researchers are seeking to cultivate cotton plants with longer fibers. In this study, cotton fiber genes were systematically reviewed through meta-analysis in terms of extending and shortening fiber and the use of different research technologies for the first time. PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), and Baidu Xueshu databases were included as literature retrieval sources. A total of 21,467 articles were retrieved, and 45 articles were used in the final analysis. Data analysis was performed using RevMan 5.4 software. To shorten cotton fiber length, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology was superior to virus-induced gene silencing (VIGS) technology and RNA interference (RNAi) technology [p = 0.002, MD = −1.05, 95% CI (−1.73, −0.37), Chi² = 39.89]. To increase cotton fiber length, CRISPR-Cas9 technology had a similar effect as VIGS technology [p = 0.12, MD = −0.59, 95% CI (−1.33, −0.15), Chi² = 0.17]. When some genes (GhLAC15, GhALDH7B4, GhMDHAR1A/GhDHAR2A, STTM-miR396b, GhMYB44, GhFP2, GhMYB7, GhKNL1, GhTCP4, GhHDA5, GhGalT1, GhKNOX6, GhXB38D, and GhBZR3) were damaged, cotton fiber length increased. Furthermore, we found that after gene interference, the fiber-shortening genes occurred more frequently than the fiber-elongating genes. Synergistic research on these genes may better promote cotton fiber elongation.