Fine tuning wheat heading time through genome editing of transcription factor binding sites in Ppd-1 gene promoter.

Increasing the productivity and adaptability of agricultural plants often depends on optimizing heading time. To develop common wheat lines with accelerated heading and investigate its regulation, we targeted the PPD-1 genes, which control photoperiod sensitivity. Large deletions in the promoter regions of these genes are known to disrupt their expression, resulting in early heading. Using CRISPR/Cas9 genome editing, we generated wheat plants with mutations in the promoter regions of the Ppd-D1 and Ppd-B1 genes. These mutations included nucleotide substitutions, deletions, and insertions ranging from several to hundreds of base pairs, occurring within probable transcription factor binding sites, that may influence gene expression. Under short-day conditions, we assessed PPD-1 gene expression in T0 plants and T2 lines with different mutations. Our analysis revealed that deletions spanning the CHE transcription repressor binding sites altered gene expression patterns, supporting the hypothesis regarding the role of these cis-elements in regulating PPD-1 expression. Furthermore, plants with different mutations displayed distinct diurnal expression patterns, suggesting the involvement of additional transcription factors in the regulation of this gene. Evaluation of heading time in T1 and T2 families with different mutations demonstrated that plants with mutations affecting the "core region", including the CHE binding sites, initiated heading significantly earlier than those without mutations.