Multi-Level Bioinformatics Analysis Reveals Expression Divergence and Potential Transcriptional Regulation of the PSY Gene Family in Marigold

Marigold (Tagetes erecta L.) petals accumulate high levels of lutein and other carotenoids, making marigold an important plant source of natural lutein. Phytoene synthase (PSY) is a key rate-limiting enzyme in the carotenoid biosynthetic pathway, yet the expression divergence of the PSY family and its upstream transcriptional regulation in marigold remain unclear. In this study, we identified four PSY genes, TePSY1-TePSY4, in the re-annotated marigold genome. Transcriptome analysis revealed pronounced expression divergence among the four TePSY genes. TePSY1 showed the highest overall expression among the four genes in the analyzed transcriptomic datasets. TePSY2 and TePSY3 maintained relatively stable expression patterns. TePSY4 exhibited low overall expression but marked increases in a subset of samples, highlighting distinct expression patterns across the integrated transcriptomic datasets. Using random forest models, we identified the ethylene-responsive factor (ERF) family member Chr_04-g1253.t1 as the most important transcription factor associated with the TePSY1 module. PTF-Vāc prediction further identified five candidate binding motifs for Chr_04-g1253.t1 within the TePSY1 upstream region. AlphaFold3-based protein–DNA complex modeling provided additional structural evidence consistent with their potential interaction. This study thus reveals divergent evolutionary, expression, and potential transcriptional regulatory patterns among the marigold PSY family members through integrated multi-level computational analyses. In addition, these findings provide candidate regulatory relationships and a framework for investigating the transcriptional regulation of PSY genes in marigold.