NADPH oxidases (NOXs), mostly known as respiratory burst oxidase homologs (RBOHs), are the key producers of reactive oxygen species (ROS) in plants. A lot of literature has addressed ROS signaling in plant development regulation and stress responses as well as on the enzyme’s structure, evolution, function, regulation and associated mechanisms, manifesting the role of NOXs/RBOHs as the vital performers and center hubs during plant growth and signaling. This review focuses on recent advances of NOXs/RBOHs on cell growth, hormone interaction, calcium signaling, abiotic stress responses, and immunity. Several primary particles, including Ca²⁺, CDPKs, BIK1, ROPs/RACs, CERK, FER, ANX, SnRK and SIK1-mediated regulatory mechanisms, are fully summarized to illustrate the signaling behavior of NOXs/RBOHs and their sophisticated and dexterous crosstalks. Diverse expression and activation regulation models endow NOXs/RBOHs powerful and versatile functions in plants to maintain innate immune homeostasis and development integrity. NOXs/RBOHs and their related regulatory items are the ideal targets for crop improvement in both yield and quality during agricultural practices.

Hexaploid wheat has a large genome, making it difficult for transgenes to produce phenotypes due to gene redundancy and tight linkage among genes. Multiple gene copies typically necessitate multiple targeting events during gene editing, followed by several generations of self-crossing to achieve homozygous genotypes. The high cost of transgenesis in wheat is another issue, which hinders the easy availability of gene-edited materials in wheat. In this study, we developed a comprehensive approach to improve wheat gene editing efficiency. First, we established a protoplast-based system to evaluate the relative efficiency of gene editing targets, which enabled the rapid and effective selection of optimal sgRNAs. We then compared two transformation strategies: biolistic bombardment and Agrobacterium-mediated transformation for generating edited wheat lines. Although biolistic bombardment showed higher initial editing efficiency, Agrobacterium-mediated transformation proved more effective for obtaining homozygous mutants. Notably, we discovered that deploying the same sgRNA through different vectors enhanced editing efficiency, whereas overlapping but distinct sgRNAs exhibited interference effects. Finally, we optimized the VITF-edit (virus-induced transgene free editing) technique using BSMV delivery to establish a relatively simple and easily applied wheat gene editing method for general laboratories.