Knockout of OsRbohD (the NADPH Oxidase Gene) Enhances Saline-Alkaline Stress Tolerance and Grain Yield in Rice by...

Saline-alkaline (SA) stress, characterized by high salinity and alkalinity, severely constrains global crop productivity by inducing excessive accumulation of reactive oxygen species (ROS). This study revealed that CRISPR/Cas9-mediated knockout of the NADPH oxidase gene OsRbohD (OsRbohD-KO) significantly enhanced SA stress tolerance in rice. Compared with wild-type plants, the OsRbohD-KO lines exhibited a marked reduction in ROS accumulation in seeds, leaves, and roots under both saline and alkaline stress. This resulted in significantly improved seed germination, bud development, and root elongation. Physiologically, the OsRbohD-KO seedlings maintained higher leaf survival, chlorophyll content and K⁺ content, while showing reduced malondialdehyde (MDA) levels, membrane injury and Na⁺ accumulation. In severely SA soils, OsRbohD-KO increased seedling emergence by 5.77-28.85%, plant growth by 1.51-23.64%, root growth by 6.80-23.66%, and grain yield by 12.53-175.23%, driven by improvements in spikelets per panicle, grain filling, and 1000-grain weight. Molecular analysis revealed that OsRbohD-KO upregulated the expression of stress-related genes, including a cell death inhibitor gene (OsBI-1), Na⁺ and K⁺ transporter-related genes (OsHKT1, OsHKT7 and OsPEX11), proline biosynthesis-related genes (OsP5CS1 and OsP5CS2), and a cell protection and stress signal transduction-related gene (OsJRL), and downregulated the expression of the cell death gene OsKOD-1. Conversely, OsRbohD overexpression lines showed increased sensitivity to SA stress. Our results suggest that suppressing OsRbohD is a highly effective strategy for alleviating SA damage and improving rice productivity in marginal environments.