Abstract
Increasing grain yield is the primary goal of crop improvement, which is globally affected by the low availability of soil phosphate (Pi). Overexpressing Pi transporters to enhance Pi uptake often results in Pi toxicity and growth retardation. Despite advances in genetic engineering, targeting the cis-regulatory motifs of Pi transporters remains underexplored for understanding plant mechanisms and improving Pi status. Here, we demonstrate that the excision of the transcription inhibitor motif from the promoter of the Pi transporter OsPHO1;2 enhances its expression and increases root-to-shoot Pi transport, leading to improved grain yield. Through in silico and DNA-protein interaction studies, we show the role of the OsWRKY6 transcription factor in negatively regulating OsPHO1;2 expression by binding to the cis-regulatory element (W-box) present in its promoter. The oswrky6 knockout lines exhibit higher OsPHO1;2 expression and improved shoot Pi levels. Furthermore, we engineered the OsPHO1;2 promoter to precisely remove the W-box and enhance OsPHO1;2 expression. Phenotypic and physiological evaluations at the vegetative stage indicate that OsPHO1;2 promoter-edited (OsPHO1;2:PE) lines have increased shoot length, plant biomass and greater root-to-shoot Pi export under both low and normal P conditions. Notably, the (33)P uptake assay reveals that OsPHO1;2:PE lines display enhanced root Pi uptake, supported by higher expression of root-associated Pi transporters (OsPHTs). An extensive agronomic assessment shows that OsPHO1;2:PE lines achieve increased seed and panicle numbers, thereby raising yield without affecting seed quality. Our findings provide valuable insights into the potential of promoter editing to improve Pi use and enhance crop yield.