Editing cis-elements of OsPHO1;2 improved phosphate transport and yield in rice.

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