Bioinformatic analysis, gene expression, and in silico characterization of the transcription factor MYB1 in sweet orange (Citrus sinensis)

The diverse MYB transcription factor family regulates key plant processes, including pigment biosynthesis and critical abiotic stress responses such as cold tolerance. This study aims to characterize the MYB1 protein and analyze its expression in citrus species subjected to low-temperature stress. One-year-old grafted seedlings of four citrus cultivars on sour orange rootstock were subjected to controlled freeze stress in a factorial, completely randomized design. Temperatures were gradually decreased to + 4 °C (control), − 4 °C, and − 8 °C for 10 h, with root zones insulated. Following a 24-hour recovery at + 4 °C, leaf samples were collected. Anthocyanin content was quantified spectrophotometrically from acidified methanol extracts. Total RNA was extracted from frozen leaf powder, treated with DNase I, and reverse-transcribed into cDNA. Gene-specific primers for MYB1 and the EF1 reference gene were designed using Oligo7 software. Quantitative PCR was performed using SYBR Green master mix. Gene expression was calculated via the ΔΔCt method, and data were analyzed by ANOVA and Duncan’s test ( p < 0.05). ANOVA revealed a highly significant interaction between temperature and cultivar for all traits. Anthocyanin content increased with declining temperature in a genotype-dependent manner, peaking in Thomson Navel orange at − 8 °C. MYB1 gene expression was significantly upregulated by cold stress, with the highest induction in Thomson Navel orange (36.25-fold at − 8 °C). Bioinformatic analysis characterized the MYB1 gene, its promoter containing stress-responsive elements, and predicted the protein’s structure, which was validated as acceptable by Ramachandran plot analysis. Cold stress significantly induces MYB1 expression and anthocyanin accumulation in citrus, with strong genotype-specific variation.