Screening of MYB1R1 interaction with promoter to regulate anthocyanin biosynthesis in peaches.

BACKGROUND

The floral color variegation of cultivar 'Sahong Tao' is distinctive and possesses significant ornamental value. Currently, there are no relevant reports on how MYB transcription factors (TFs) interact with promoter to regulate the flower color variegation in peach.

METHODS

In this study, we screened for proteins that interact with the promoter using yeast one-hybrid (Y1H) and next-generation sequencing (NGS). The NGS data were aligned with the database (TAIR10) utilizing Python 3.10.4. PlantTFDB was employed to identify TFs, while PlantRegMap was used to predict TFs that interact with the promoter. The Y1H assay verified MYB1R1 interaction with promoter, and Y1H-AOS predicted their binding sites. The physicochemical properties, structure and interacting proteins of MYB1R1 were analyzed using bioinformatics methods. Sequence alignment and phylogenetic tree analyses of were performed. Finally, the tissue expression specificity of and in 'Sahong Tao' was examined using qRT-PCR.

RESULTS

The Y1H and NGS results indicate that 1,190 proteins interact with the 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 , 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 promoter. Y1H-AOS was used to confirm 24 interaction binding sites. 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 and . Promoter prediction analysis for reveals multiple hormone- and stress-related -acting elements. MYB1R1 may interact with bHLH and other proteins to perform its functions. In variegated petals, expression is higher and 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 and the regulatory mechanism of MYB- in the flower color of 'Sahong Tao'.