Screening of MYB1R1 interaction with <i>LDOX</i> promoter to regulate anthocyanin biosynthesis in peaches

The Y1H and NGS results indicate that 1,190 proteins interact with the <i>LDOX</i> promoter. Among these, 20 TFs were identified, including ERF, MYB, NF-YB, SBP, S1Fa-like, TCP, bHLH, LBD, ZF-HD, C3H, DBB, MYB-related, and HD-ZIP. Of the 1,190 proteins, 1,146 exhibit high similarity to homologs in <i>Arabidopsis</i>, with 332 classified as RNA binding proteins and 124 as DNA binding proteins. A comparison with the NGS results identified seven TFs that align with predictions from PlantRegMap. Based on these findings, we selected MYB44 (PRUPE_6G229000, PRUPE_1G430000) and MYB1R1 (PRUPE_5G182000) as candidate members. Y1H assays demonstrated that MYB1R1 interacts with the <i>LDOX</i> promoter. Y1H-AOS was used to confirm 24 interaction binding sites. <i>MYB1R1</i> consists of an 897 bp full-length CDS, encoding 298 amino acids, with a predicted molecular weight of 32.49 kDa and a theoretical isoelectric point of 7.20. MYB1R1 features a typical SANT-MYB domain, and its secondary structure is predominantly composed of irregular coils. Phylogenetic analysis indicates a close evolutionary relationship between MYB1R1 from 'Sahong Tao' and both <i>Prunus avium</i> and <i>Prunus speciosa</i>. Promoter prediction analysis for <i>MYB1R1</i> reveals multiple hormone- and stress-related <i>cis</i>-acting elements. MYB1R1 may interact with bHLH and other proteins to perform its functions. In variegated petals, <i>MYB1R1</i> expression is higher and <i>LDOX</i> expression is lower compared to red petals, suggesting that MYB1R1 negatively regulates anthocyanin synthesis by interacting with LDOX. This study contributes to elucidating the function of <i>MYB1R1</i> and the regulatory mechanism of MYB- <i>LDOX</i> in the flower color of 'Sahong Tao'.