ChIP-seq Identification and Functional Characterization of Pea NTDPase DNA-Binding Sites for in Promoters of Arabidopsis Genes

Arabidopsis seedlings (Col-0 background) over-expressing a 4Myc-tagged pea NTPDase psNTP9 (PS) or psNTP9-DM (double mutant, DM; S208L, P216R mutations) were grown for 7 days in continuous light on standard 1/2x MS medium. The DM mutations are located in a potential calmodulin-binding domain that overlaps with the nuclear localization signal. These mutations were found to reduce nuclear targeting and alter DNA-binding of DM, relative to PS protein. PS and DM exhibited largely non-overlapping sets of binding sites in promoters, and distinct expression profiles for potentially-regulated target genes in yeast. (Tripathy et al. 2025 Front. Plant Sci.16:1584871). In the present study, PS and DM binding sites in Arabidopsis were identified and functional studies in vitro (EMSA) were employed to further characterize PS and DM binding to enriched TF motifs in both yeast and plant promoters.