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

Increasing grain yield is the main aim of crop improvement, which is globally impacted by the low availability of soil phosphate (Pi). Overexpressing Pi transporters for increasing Pi uptake often result in Pi toxicity and growth retardation. Despite advancements in genetic engineering, targeting cis-regulatory motifs of Pi transporters remains underexplored for understanding plant mechanisms and improving Pi status. Here, we demonstrated that the excision of the transcription inhibitor motif from the promoter of Pi transporter, OsPHO1;2, enhanced its expression and increased root-to-shoot Pi transport, leading to improved grain yield. Using, in-silico and DNA-protein interaction studies, we show the role of OsWRKY6 transcription factor in the negative regulation of OsPHO1;2 expression by binding to the cis-regulatory element (W-box) present in its promoter. oswrky6 knockout lines displayed a higher OsPHO1;2 expressions, and improved shoot Pi levels. Further, we engineered the OsPHO1;2 promoter for precisely removing the W-box and enhancing OsPHO1;2 expression. The phenotypic and physiological evaluation at the vegetative stage shows that OsPHO1;2 promoter edited (OsPHO1;2:PE) lines have increased shoot length, plant biomass and more root-shoot Pi export under low and normal P conditions. Notably, the 33Pi uptake assay reveals that OsPHO1;2:PE lines exhibited enhanced root Pi uptake, supported by higher root-associated Pi transporters (OsPHTs) expression. An extensive agronomical assessment revealed that OsPHO1;2:PE lines acquire increased seed and panicle numbers, thereby increasing yield without affecting seed quality. Our findings offer valuable insight into the potential of promoter editing to raise plants with better Pi use and improved crop yield.